CN109975086A - GHH absorbs effect and the verification method of EE2 and Cd to rye grass - Google Patents
GHH absorbs effect and the verification method of EE2 and Cd to rye grass Download PDFInfo
- Publication number
- CN109975086A CN109975086A CN201811114767.1A CN201811114767A CN109975086A CN 109975086 A CN109975086 A CN 109975086A CN 201811114767 A CN201811114767 A CN 201811114767A CN 109975086 A CN109975086 A CN 109975086A
- Authority
- CN
- China
- Prior art keywords
- ghh
- soil
- rye grass
- plant
- added
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/3103—Atomic absorption analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
Landscapes
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a kind of GHH to absorb effect and the verification method of EE2 and Cd to rye grass, and EE2-Cd combined contamination soil can be repaired by verifying to obtain rye grass joint GHH with verification method of the invention；GHH is the Hyphomicrobium of deposit number CCTCC NO:M2017539, is named as Hyphomicrobium sp.GHH；Inoculation GHH significantly promotes the growth of plant, improves rye grass to the extraction efficiency of EE2 and Cd；In grown on soil rye grass and GHH is inoculated with to urease activity and microbial biomass carbon content with facilitation, can improve soil environment.
Description
Technical field
The present invention relates to microbial environment administer field, especially a kind of GHH to rye grass absorb EE2 and Cd effect and
Verification method.
Background technique
Environment incretion interferent (EEDCs) and its influence to human and animal's endocrine system are just by more and more
More concerns.Ethinylestradiol is as typical environment incretion interferent and the active constituent of many contraceptives.While it
Also it is applied to the treatment for the diseases such as prostate cancer and breast cancer, climacteric and post-menopause syndrome, sclerotin be loose.
Ethinylestradiol enters environment via human body or animal excrements.Farmland is the main source of grain, and irrigate,
Runoff and feces of livestock and poultry etc. can all make ethinylestradiol continue into farmland.Some researches show that ethinylestradiol can be rapid
It is adsorbed in soils and sediments.In addition, ethinylestradiol can be enriched in the root of the plants such as plant such as kidney bean, celery, and
It is transported in leaf by root.It can be seen that the ethinylestradiol in agricultural land soil is by direct Pollution Plant, while also can be with food
The mode harmful to human and animal health of chain.
Easily in conjunction with estrogen receptor, estrogenic potency is day for human body and the studies have shown that ethinylestradiol of fish
2-30 times of right estrogen.The ethinylestradiol of low concentration (ng/L) is sexual gland and the gender growth that can inhibit fish, destroys it
Endocrine system influences its secondary feature.At present in sewage treatment plant, artificial swamp, soil, river, underground water and aqueous
There is the residual for detecting ethinylestradiol in surface sediments, seawater and marine sediment.Ethinylestradiol is to human health, life
The influence of object and ecology attracts people's attention, and harm also constitutes hidden danger to human survival and Environmental security.
The mode of processing ethinylestradiol has the methods of absorption, photodissociation, chemical oxidation, MBR and biotechnology at present.Object
Change processing technique to be mainly used in the pretreatment of sewage treatment plant and the purifier of water purifier, this method is used for soil remediation
Rehabilitation cost is high when middle, repair coverage limitation.Biological prosthetic have many advantages, such as small investment, high-efficient, is renovation of organic pollution soil
The research hotspot of earth.
Cadmium (Cd) is mainly used for the plating of steel, iron, copper, brass and other metals, strong to the anti-corrosion capability of alkaline matter.
Cadmium can be used for manufacturing the small in size and big battery of capacitance.The compound of cadmium is also largely used to production pigment and fluorescent powder.Vulcanization
Cadmium, cadmium selenide, cadmium telluride are for manufacturing photocell.Because use scope is wide, the garbage waste with cadmium is also very much, cadmium
Be more toxic, it is serious by the air of cadmium pollution and food harm to the human body and slower in human body metabolism, Japan because of cadmium poisoning once
Occur " itai-itai ".
The method of prior art coal multi-purpose greatly or charcoal reduction or sulfuric acid leaching and zinc dust precipitation is from flue dust or cadmium containing cadmium
Cadmium metal is obtained in slag, but the existing cadmium in soil, the case where such large area dispersed distribution, be just difficult to usual side
Method, aggregation are extracted.
Market need a kind of pollution-free, nuisanceless, environment amenable processing technique can absorb the EE2 in soil and
Cadmium, the present invention solve such problems by microorganism and plant cooperation.
Summary of the invention
To solve the deficiencies in the prior art, it is an object of the invention to
In order to achieve the above objectives, the present invention adopts the following technical scheme that:
GHH absorbs the effect of EE2 and Cd to rye grass, comprising: rye grass joint GHH can repair EE2-Cd combined pollution
Soil；GHH is the Hyphomicrobium of deposit number CCTCC NO:M2017539, is named as Hyphomicrobium sp.GHH.
The effect that GHH above-mentioned absorbs EE2 and Cd to rye grass is inoculated with GHH's with the raising of Cd concentration in soil
The removal rate of EE2 and Cd reduces in Soil As Influenced By Ryegrass Plants, and extracted amount increases.
GHH above-mentioned absorbs the effect of EE2 and Cd to rye grass, and inoculation GHH can promote the growth of rye grass, improve black
Extraction efficiency of the wheat straw to EE2 and Cd.
GHH above-mentioned absorbs the effect of EE2 and Cd to rye grass, and at germination the 3rd day of rye grass, every basin was added
The GHH bacteria suspension of 10mL is added in soil, and the 14th day when is added primary.
GHH above-mentioned absorbs the effect of EE2 and Cd, the OD of GHH bacteria suspension to rye grass600Value is 1.
GHH above-mentioned absorbs the effect of EE2 and Cd to rye grass, in grown on soil rye grass and is inoculated with GHH to urase
Activity and microbial biomass carbon content have facilitation.
GHH absorbs the verification method of the effect of EE2 and Cd to rye grass, includes the following steps:
Prepare material: configuration contains EE2, and the soil of Cd plants rye grass, is inoculated with GHH；GHH is deposit number CCTCC
The Hyphomicrobium of NO:M2017539 is named as Hyphomicrobium sp.GHH；
Processing group is arranged: 4 processing groups of setting are respectively as follows: the blank group that do not add GHH and do not plant rye grass, individually
GHH is added, rye grass is individually planted, plant rye grass and adds GHH, each processing group is all provided with 3 in parallel, germinates in rye grass
28d afterwards, harvesting sampling, measures indices；
It measures phytomass: after harvesting plant cleaning, measuring root long, dry measured weight；
Measure the content of EE2 in plant and soil；
Measure the content of Cd in plant and soil；
Measure urease activity and microbial biomass carbon in soil.
GHH above-mentioned absorbs the verification method of the effect of EE2 and Cd to rye grass, and EE2's contains in measurement plant and soil
The specific method of amount includes:
EE2 in extraction and determination plant: fresh plant sample is ground uniformly in liquid nitrogen, takes stem sample or root sample to be placed in poly-
In propylene centrifuge tube, deionized water and Acidifying acetonitrile is added, is vibrated and is mixed with turbine mixer, it is then ultrasonic, salt extraction is added
Agent, and vibrated immediately with turbine mixer, after mixed liquor centrifugation, 1~2h is placed in freezing, takes supernatant to be filtered with membrane filtration, filtrate
Methanol constant volume is added after nitrogen drying is dry and is measured；The salt extraction agent includes: MgSO4, NaCl, Na3C6H5O7,
C6H5Na2O7；
EE2 in extraction and determination soil: soil sampling grinds uniformly in liquid nitrogen, stem sample or root sample is taken to be placed in polypropylene centrifuge
Deionized water and Acidifying acetonitrile is added in Guan Zhong, is vibrated and is mixed with turbine mixer, then ultrasonic, salt extraction agent is added, and immediately
It is vibrated with turbine mixer, after mixed liquor centrifugation, supernatant is transferred to containing anhydrous MgSO4And C18Polypropylene centrifuge tube
In；After vortex centrifugal, takes supernatant progress nitrogen drying dry, add methanol constant volume；The salt extraction agent includes: MgSO4, CH3COONa；
The measuring method of EE2: dense using the EE2 in high performance liquid chromatography and chromatogram column analysis plant or soil extract object
Degree.
GHH above-mentioned absorbs the verification method of the effect of EE2 and Cd to rye grass, measures the content of Cd in plant and soil
Specific method include:
Cd content in measurement plant: plant is cleaned and is divided into root and aerial part, shreds to be put into baking oven and dry, will dry
After plant sample after dry is crushed with pulverizer, it is sieved to be measured；Plant root or aerial part are weighed respectively, add HC1O4With
HN03, disappeared to boil tank and disappear to boil to solution with polytetrafluoroethylene (PTFE) and be clarified completely；Disappear after boiling, the boil liquid that disappears membrane filtration, by filtrate
It is transferred in volumetric flask, constant volume；
Cd content in measurement soil: taking pedotheque, and after sieving, HNO is added in natural air drying3, HCIO4And HF, disappear boil to
Clarification completely；The boil liquid that disappears membrane filtration, filtrate is transferred in volumetric flask, constant volume；
With Cd content in atomic absorption spectrometry plant and soil.
GHH above-mentioned absorbs the verification method of the effect of EE2 and Cd to rye grass,
The specific method of urease activity includes: in measurement soil
Air-dried soil sample is taken, is placed in conical flask, toluene is added and mixes, the urea liquid and Citrate buffer added after standing
Sample is placed in incubator and sufficiently shakes up, mixture is centrifuged by liquid, takes supernatant and is diluted with distilled water, will be diluted
Supernatant is transferred in centrifuge tube, adds sodium phenate and sodium hypochlorite, and sample is mixed well, and is stood, is diluted with distilled water, is used
Ultraviolet-spectrophotometric measures absorbance, determines NH in sample according to standard curve3The concentration of-N, urease activity U=y × 10 × V
× W × T, wherein y NH3The concentration of-N；10 be thinner ratio；V is reaction system total volume；W is example weight；When T is reaction
Between；
Measurement Soil Microorganism amount carbon content specific method include:
Moist soil is weighed, is put into conical flask, arginine aqueous solution is added, sample is placed and is cultivated, taking-up is put into ice
It in case, places, then sample taking-up is placed on and is thawed at room temperature, KCl aqueous solution is added in the completely backward solution that thaws, concussion mentions
It takes, solution is placed in centrifuge tube and is centrifugated, supernatant membrane filtration measures filtrate；Ammonium content indophenols in filtrate
Blue colorimetric method for determining；As a result it is calculated as ω (C)=0.01 × NH4 +-Nmg·kg-1, in formula: ω (C) is microbes biomass carbon
Bc mass fraction, mgkg-1；0.01 is converted to the coefficient of Bc for arginine induction ammoniation process.
The invention has the beneficial effects that:
EE2-Cd combined pollution soil can effectively be repaired by verifying to obtain rye grass joint GHH with verification method of the invention
Earth；
Inoculation GHH significantly promotes the growth of plant, improves rye grass to the extraction efficiency of EE2 and Cd；
When Cd concentration is respectively 5mg/kg, reparation 28d is best to the removal effect of EE2 in soil and Cd, the removal of EE2
Rate is respectively 89%；The removal rate of Cd is 0.81%；
In grown on soil rye grass and GHH is inoculated with to urease activity and microbial biomass carbon content with facilitation, energy
Enough improve soil environment.
Detailed description of the invention
Fig. 1 is the column diagram of verification method of the present invention root long of rye grass in 28d；
Fig. 2 is the column diagram of verification method of the present invention dry weight of the aerial part of rye grass in 28d；
Fig. 3 is the column diagram of verification method of the present invention EE2 content of the root of rye grass in 28d；
Fig. 4 is the column diagram of verification method of the present invention EE2 content of the aerial part of rye grass in 28d；
Fig. 5 is the column diagram of verification method of the present invention Cd content of the root of rye grass in 28d；
Fig. 6 is the column diagram of verification method of the present invention Cd content of the aerial part of rye grass in 28d；
Fig. 7 is the column diagram of verification method of the present invention removal rate of soil EE2 in 28d；
Fig. 8 is the column diagram of verification method of the present invention removal rate of soil Cd in 28d；
Fig. 9 is the column diagram of verification method of the present invention urease activity in soil in 28d；
Figure 10 is the column diagram of verification method of the present invention MBC content in soil in 28d.
Specific embodiment
Specific introduce is made to the present invention below in conjunction with the drawings and specific embodiments.
GHH is the Hyphomicrobium of deposit number CCTCC NO:M2017539, is named as Hyphomicrobium sp.GHH, is protected
Hide unit: China typical culture collection center；Address: Wuhan, China Wuhan University；Preservation date: on September 25th, 2017.
1. materials and methods
1.1. experimental material
Suburb Areas of Hangzhou farmland is picked up from for examination soil, removes plant and animal residues and stone, natural air drying, mistake after soil collection
It is mixed well after 2mm sieve.Physiochemical properties of soil is measured by soil agrochemistry traditional analysis method[20].Heavy metal content in soil and
Basic physical and chemical: pH 5.6, cation exchange capacity (CEC) 9.3cmol/kg, the content of organic matter 4.1%, alkaline hydrolysis nitrogen content 413.2mg/
Kg, available phosphorus content 28.2mg/kg, effective potassium content 216.7mg/kg, total Cd content 0.09mg/kg, EE2 are not detected.Addition
EE2 is simultaneously mixed well, and the contaminated soil that EE2 external source concentration is 25mg/kg is made.By configured Cd (NO3)2Aqueous solution is added
In soil containing 25 mg/kg EE2, mixes well, Cd is respectively prepared2+External source concentration be 0mg/kg, 5mg/kg, 20mg/kg,
The contaminated soil of 80mg/kg.The 60% of field capacity is added water to, is used to test after stablizing 30d.Every basin (diameter 18.5cm ×
High 21cm) dress soil 2kg (dry weight), basin underlay pallet.
The implantation methods of rye grass: the ryegrass seed for choosing full grains is immersed in 3% hydrogen peroxide solution, disinfection
30min, with sowing after distilled water flushing, every basin sows 250 seeds, guarantees every basin > 200 plant plant after germination.Rye grass exists
Zhejiang Prov Industrial And Commercial University's Environmental Science and Engineering institute laboratory building is cultivated in greenhouse, and Greenhouse day/evening hours are set as 16h/8h, white
It/night temperatures control at 25 DEG C/15 DEG C, regular watering makes soil moisture be maintained at the 60% of field capacity.
The inoculation method of EE2 degradation bacteria GHH: by isolated GHH in shaking table with 120rpm in MSM culture solution (NH4Cl
2g/L, KH2PO40.5g/L, K2HPO40.5g/L, MgSO4·7H2O 0.2g/L, FeSO4·7H2O 0.01g/L, CaCl2
0.03g/L, MnSO4·H2O 0.001g/L, ZnSO40.001g/L, pH 7) in culture for 24 hours, 8000rpm be centrifuged 10min, sink
Shallow lake is washed twice with 0.85% sterile NaCl, is then resuspended with deionized water to OD600Value is 1, as GHH inoculum.In seed
Germinate 3d, is added in soil with every basin 10mL inoculum, and when 14d is added primary.
1.2. processing group is arranged
4 processing groups are arranged in test altogether, are respectively as follows: (1) and do not add GHH and do not plant the blank group (C) of rye grass, and 2) it is single
Rye grass (P) is individually planted in solely addition GHH (B), (3), and (4) plantation rye grass is simultaneously added GHH (B+P), and every processing group is all provided with 3
In parallel.28d after rye grass germination, harvesting sampling, measures indices.
The measurement of 1.3 phytomass
Distilled water repeated flushing plant is used after plant harvesting, then with ultrapure water 3~4 times, clean plant is inhaled
Water paper blots plant surface moisture and measures root long.By aerial part in 105 DEG C of water-removing 2h, then in 80 DEG C of drying until permanent
Weight weighs aerial part dry weight.Remainder is put into low-temperature preservation in hermetic bag and is used for subsequent measurements.
The measurement of EE2 content in 1.4 plants and soil
The method that the extraction of EE2 refers to Flores in plant[21].Fresh plant sample is ground in liquid nitrogen uniformly.
It takes stem sample 3g or root sample 1g to be placed in 50mL polypropylene centrifuge tube, 10mL deionized water is added and 10mL Acidifying acetonitrile (contains 1%
Acetic acid), mixing 1min is vibrated with turbine mixer, then ultrasound 15min.Salt extraction agent (4g MgSO is added4, 1g NaCl,
1g Na3C6H5O7With 0.5g C6H5Na2O7), and 1min is vibrated with turbine mixer immediately.Mixed liquor is centrifuged with 5000rpm
It takes the supernatant of 6~8mL to be filtered with 0.22 μm of membrane filtration after 10min, -20 DEG C of 1~2h of placement, 1 mL is added after the drying of filtrate nitrogen is dry
Methanol constant volume is simultaneously measured.
The extraction reference of EE2 in soil[22]Method.Soil sampling 2g, extracting method is similar to plant, and difference is: soil
The salt extraction agent of sample is 4g MgSO4With 1.5g CH3COONa；About 8mL supernatant is transferred to after centrifugation anhydrous containing 900mg
MgSO 4With 60mg C1815mL polypropylene centrifuge tube in；After vortex centrifugal, takes the progress nitrogen drying of 6mL supernatant dry, add 1mL
Methanol constant volume.
The measuring method of EE2: high performance liquid chromatography (HPLC, Agilent 1260, USA) and chromatographic column (AORBAX are used
Eclipse XDB-C18,5 μm, 4.6mm × 150mm) EE2 concentration in analysis plant or soil extract object.HPLC operating condition
Are as follows: mobile phase acetonitrile: water=45:55, Detection wavelength 280nm, flow velocity 0.8mL/min, 30 DEG C of column temperature, 20 μ L of sample volume.
The measurement of Cd content in 1.5 plants and soil
Measuring method of the measurement of Cd with reference to Zhu et al.[14].The measurement of Cd content in plant: plant is cleaned and is divided into root
Portion and aerial part are shredded to be put into baking oven and be dried, after the plant sample after drying is crushed with pulverizer, sieve with 100 mesh sieve to
It surveys.The plant root or aerial part for weighing about 0.1g respectively, add 1.0mL HC1O4With the dense HN0 of 4.0mL3, use polytetrafluoroethylene (PTFE)
Disappear and boil tank at 160 DEG C, disappears to boil to solution and clarify completely；Disappear after boiling, 0.22 μm of membrane filtration of the boil liquid that disappears turns filtrate
Enter in 50mL volumetric flask, constant volume.
The measurement of Cd content in soil: taking pedotheque with soil sampler, is placed in ventilation natural air drying, crosses 2mm sieve；It weighs
The dense HNO of 1.0mL is added in 0.3g pedotheque3, 1.0mL HCIO4With 1.0mL HF, disappears at 180 DEG C and boil to complete clarification；Disappear
After boiling, filtrate is transferred in 50mL volumetric flask, constant volume by 0.22 μm of membrane filtration of the boil liquid that disappears.Use Atomic Absorption Spectrometer
(ICE 3300, Thermo Scientific) measures Cd content in plant and soil.
The measurement of urease activity and microbial biomass carbon (MBC) in 1.6 soil
Urease activity measurement is with reference to Guo etc.[23]Method: take 5g to air-dry soil sample, be placed in 50mL conical flask, be added 1mL
Toluene mixes, and 6.7 citrate buffer of urea liquid and 20mL pH of 10mL 10% is added after standing 15min.Sample is set
It is shaken up for 24 hours and sufficiently in 37 DEG C of incubators.Then, mixture is centrifuged 10min with 10000rpm.Take supernatant and with distillation
Water dilutes 10 times.The 400 diluted supernatants of μ L are transferred in 2mL centrifuge tube, L chlorine of sodium phenate and 60 μ of 80 μ L1.35M is added
Sour sodium (Active Chlorine 0.9%).Sample is mixed well, 20min is stood, is diluted with 460 μ L distilled water.Use ultraviolet-light splitting light
Degree meter (Thermo Genesys 10, USA) surveys absorbance at 630nm, determines NH in sample according to standard curve3- N's is dense
Degree.Urease activity U=y × 10 × V × W × T, wherein y is NH3The concentration of-N；10 be thinner ratio；V is that reaction system is overall
Product: 2mL；W is example weight: 0.25g；T is the reaction time: 1d.
Arginine-indophenol blue colorimetry of the measurement of microbial biomass C (MBC) with reference to Lu Kunru et al.[24].MBC's
Measurement: weighing the moist soil for being equivalent to that dry weight is 10g, be put into the taper of 250mL, and every gram of soil sample is added arginine content and is
The arginine aqueous solution (the ratio 1:1 for making soil and water) of 0.3mg, sample is placed at 25 DEG C and is cultivated, and is taken out and is put after 2h
Enter in -15 DEG C of refrigerators, places 4h.Then sample taking-up is placed on and is thawed at room temperature, 20mL is added in the completely backward solution that thaws
The KCl aqueous solution of 2mol/L, 15min is extracted in concussion at 25 DEG C, and solution is placed in centrifuge tube and is centrifugated, and supernatant is used
0.22 μm of membrane filtration measures rapidly filtrate.Ammonium content is measured with indophenol blue colorimetry in filtrate.As a result it is calculated as ω (C)
=0.01 × NH4 +-N(mg·kg-1), in formula: ω (C) is microbes biomass carbon (Bc) mass fraction, mgkg-1； 0.01
The coefficient of Bc is converted to for arginine induction ammoniation process.
2. result and analysis
2.1. phytomass
As shown in Figure 1, 2, in EE2 contaminated soil, addition 5mg/kg Cd promotes the growth of rye grass, but with Cd
Concentration continues to increase, and the growth of rye grass is suppressed.Inoculation EE2 degradation bacteria GHH promotes the growth of rye grass roots, in Cd
Concentration be 0,5,20 and 80mg/kg processing in, root long respectively than not being inoculated with GHH when increase by 7.24%, 12.90%, 11.82%
With 12.28%, Aboveground Biomass of Young respectively than not being inoculated with GHH when increase by 2.99%, 0.73%, 11.29% and 11.23%.
In Fig. 1, Fig. 2: capitalization indicates the significance of difference (P < 0.05) of the different Cd concentration soil in same treatment, lowercase
Indicate the significance of difference (P < 0.05) of the identical Cd concentration soil between different disposal, similarly hereinafter.
2.2. the concentration of rye grass root and aerial part EE2
By Fig. 3,4 it is found that with Cd concentration rising, the content of rye grass root and aerial part EE2 are in that decline becomes
Gesture.When soil Cd is 80mg/kg, the EE2 content of P and B+P processing group rye grass root when 0mg/kg Cd respectively than declining
62.23% and 38.26%, aerial part EE2 content declines 60.18% and 65.74% respectively.In Cd-EE2 combined contamination soil
In, inoculation GHH significantly improves extraction of the rye grass to EE2.When soil Cd is 0,5,20 and 80mg/kg, root EE2 concentration
Increase by 3.73%, 12.40%, 35.94% and 69.56% when respectively than not being inoculated with GHH, aerial part EE2 concentration increases separately
55.76%, 71.80%, 16.71% and 35.18%.
1 rye grass of table is to EE2 BCF, TF extracted and extraction efficiency
*: capitalization indicates the significance of difference (P < 0.05) of the different Cd concentration soil in same treatment, lowercase
Indicate the significance of difference (P < 0.05) of the identical Cd concentration soil between different disposal, lower same
2.3. the concentration of rye grass root and aerial part Cd
As Fig. 5,6 with soil Cd concentration raising, rye grass root and aerial part Cd concentration significantly rise (P <
0.05).It is Cd concentration difference in rye grass root in 5,20 and 80mg/kg processing in the initial Cd concentration of soil when not being inoculated with GHH
For 16.01,50.98 and 274.76mg/kg, aerial part Cd concentration is respectively 4.32,17.19 and 73.87mg/kg.It is inoculated with GHH
Processing improves rye grass Cd concentration, and when the initial Cd concentration of soil is 5,20 and 80mg/kg, root Cd concentration is respectively than independent kind
It plants rye grass processing group and improves 88.14%, 33.81 and 7.66%, aerial part Cd concentration has been respectively increased 46.91%,
18.49% and 6.34%.
2 rye grass of table is to Cd BCF, TF extracted and extraction efficiency
2.4. in soil EE2 and Cd removal rate
As shown in fig. 7, the removal rate of soil EE2 gradually decreases with the raising of Cd concentration.When Cd concentration is 5mg/kg
When, without significant difference when the removal rate of soil EE2 is compared with 0mg/kg Cd in each processing group；It is 20 Hes in the initial Cd concentration of soil
When 80mg/kg, in C, B, P, B+P processing group soil EE2 removal rate compared with 0mg/kg Cd when decline 0% respectively, 21.43%,
6.67%, 11.11% and 35.71%, 32.86%, 18.67%, 21.11%.In the processing group of identical Cd concentration soil, EE2
Removal rate is B+P > P > B > C.
By table 2 and Fig. 8 as it can be seen that the removal effect of soil Cd is showed only as extraction of the rye grass to Cd, microorganism is not shown
Removal effect to Cd is shown.When the initial Cd concentration of soil is respectively 5,20 and 80mg/kg, the extraction of Cd in B+P processing group
Rate is respectively 0.81%, 0.43%, 0.38%, has been respectively increased 97.64%, 45.04%, 20.33% than P processing group, has shown
Inoculation GHH promotes extraction (Fig. 8) of the rye grass to Cd.With the increase of soil Cd concentration, the extraction efficiency of Cd in B+P processing group
Declined.When the initial Cd concentration of soil is 20 and 80mg/kg, in B+P processing group the recovery rate of Cd compared with 5mg/kg when distinguish
Have dropped 47.38% and 53.02%.
2.5. enzyme activity and microbial biomass carbon (MBC) in soil
As shown in fig. 6, with the increase of the initial Cd concentration of soil, urease activity first rises and declines afterwards.With the initial Cd of soil
Concentration is compared when being 0mg/kg, and in 5mg/kg, soil urease liveness improves soil Cd concentration in C, B, P and B+P processing
77.63%, 76.40%, 51.78% and 56.16%.When Cd concentration is 20mg/kg, each processing group urease activity ratio Cd is dense
Degree has dropped 38.07%, 36.00%, 34.18% and 33.72% in 5mg/kg respectively；When Cd concentration is 80mg/kg,
Compared with Cd concentration in 5mg/kg, each processing group has dropped 58.52%, 57.75%, 51.67% and 52.98% respectively.Identical Cd
In concentration soil, when being inoculated with GHH (B) with C processing group compared with without significant difference, but when plantation rye grass (P) soil urease work
Property be significantly higher than C processing group (P < 0.05), and in B+P processing the activity of soil urease in P processing without significant difference.
With the increase of the initial Cd concentration of soil, the content of MBC is first increased and is reduced afterwards, trend and Enzyme Activities in Soils one
It causes (Fig. 9,10).In identical Cd concentration soil, the content of MBC is B+P > P > B > C.It is respectively 5,20 in the initial Cd concentration of soil
When with 80mg/kg, the content of MBC increases by 19.81%, 51.72%, 56.49% than C processing respectively in B, P and B+P processing；
15.25%, 96.25%, 112.13%；49.06%, 69.81%, 145.28%.
3. conclusion
Rye grass joint GHH can effectively repair EE2-Cd combined contamination soil, be respectively 5,20 and 80mg/kg in Cd concentration
When, repair 28d to the removal effect of EE2 in soil and Cd be rye grass and GHH synergy > rye grass independent role >
GHH independent role, wherein B+P processing is respectively 89%, 80% and 71% to the removal rate of EE2 in soil；Soil Cd is gone
Except rate is respectively 0.81%, 0.43%, 0.38%.Show that rye grass joint GHH can effectively repair EE2-Cd combined pollution soil
Earth.Plantation rye grass and inoculation GHH can significantly improve urease activity and MBC content, improve soil environment, to urease activity
Facilitation with MBC content is B+P > P > B > C.Inoculation GHH significantly promotes the growth of plant, improves rye grass to EE2
With the extraction efficiency of Cd.
The basic principles, main features and advantages of the invention have been shown and described above.The technical staff of the industry should
Understand, the above embodiments do not limit the invention in any form, all obtained by the way of equivalent substitution or equivalent transformation
Technical solution is fallen within the scope of protection of the present invention.
Claims (10)
1.GHH absorbs the effect of EE2 and Cd to rye grass characterized by comprising rye grass joint GHH can repair EE2-
Cd combined contamination soil；The GHH is the Hyphomicrobium of deposit number CCTCC NO:M2017539, is named as
Hyphomicrobium sp.GHH。
2. the effect that GHH according to claim 1 absorbs EE2 and Cd to rye grass, which is characterized in that with Cd in soil
The raising of concentration, the removal rate for being inoculated with EE2 and Cd in the Soil As Influenced By Ryegrass Plants of GHH reduce, and extracted amount increases.
3. the effect that GHH according to claim 1 absorbs EE2 and Cd to rye grass, which is characterized in that inoculation GHH can
Promote the growth of rye grass, improves rye grass to the extraction efficiency of EE2 and Cd.
4. the effect that GHH according to claim 1 absorbs EE2 and Cd to rye grass, which is characterized in that in the kind of rye grass
Son germination the 3rd day, the GHH bacteria suspension that 10mL is added in every basin are added in soil, and the 14th day when is added primary.
5. the effect that GHH according to claim 4 absorbs EE2 and Cd to rye grass, which is characterized in that the GHH bacterium is outstanding
The OD of liquid600Value is 1.
6. the effect that GHH according to claim 1 absorbs EE2 and Cd to rye grass, which is characterized in that in grown on soil
Rye grass is simultaneously inoculated with GHH to urease activity and microbial biomass carbon content with facilitation.
7.GHH absorbs the verification method of the effect of EE2 and Cd to rye grass, which comprises the steps of:
Prepare material: configuration contains EE2, and the soil of Cd plants rye grass, is inoculated with GHH；The GHH is deposit number CCTCC
The Hyphomicrobium of NO:M2017539 is named as Hyphomicrobium sp.GHH；
Processing group is arranged: 4 processing groups of setting are respectively as follows: the blank group that do not add GHH and do not plant rye grass, individually add
GHH individually plants rye grass, plants rye grass and adds GHH；Each processing group is all provided with 3 in parallel, after rye grass germination
28d, harvesting sampling, measures indices；
It measures phytomass: after harvesting plant cleaning, measuring root long, dry measured weight；
Measure the content of EE2 in plant and soil；
Measure the content of Cd in plant and soil；
Measure urease activity and microbial biomass carbon in soil.
8. the verification method that GHH according to claim 7 absorbs the effect of EE2 and Cd to rye grass, which is characterized in that survey
The specific method of the content of EE2 includes: in field planting object and soil
EE2 in extraction and determination plant: fresh plant sample is ground uniformly in liquid nitrogen, stem sample or root sample is taken to be placed in polypropylene
In centrifuge tube, deionized water and Acidifying acetonitrile is added, is vibrated and is mixed with turbine mixer, it is then ultrasonic, salt extraction agent is added, and
It is vibrated immediately with turbine mixer, after mixed liquor centrifugation, 1~2h is placed in freezing, takes supernatant to be filtered with membrane filtration, the drying of filtrate nitrogen
Methanol constant volume is added after dry and is measured；The salt extraction agent includes: MgSO4, NaCl, Na3C6H5O7, C6H5Na2O7；
EE2 in extraction and determination soil: soil sampling grinds uniformly in liquid nitrogen, stem sample or root sample is taken to be placed in polypropylene centrifuge tube,
Deionized water and Acidifying acetonitrile is added, is vibrated and is mixed with turbine mixer, it is then ultrasonic, salt extraction agent is added, and use whirlpool immediately
Mixer oscillation is revolved supernatant is transferred to containing anhydrous MgSO after mixed liquor centrifugation4And C18Polypropylene centrifuge tube in；Whirlpool
After the whiz heart, takes supernatant progress nitrogen drying dry, add methanol constant volume；The salt extraction agent includes: MgSO4, CH3COONa；
The measuring method of EE2: the EE2 concentration in high performance liquid chromatography and chromatogram column analysis plant or soil extract object is used.
9. the verification method that GHH according to claim 7 absorbs the effect of EE2 and Cd to rye grass, which is characterized in that survey
The specific method of the content of Cd includes: in field planting object and soil
Cd content in measurement plant: plant is cleaned and is divided into root and aerial part, shreds to be put into baking oven and dry, after drying
Plant sample crushed with pulverizer after, be sieved it is to be measured；Plant root or aerial part are weighed respectively, add HC1O4And HN03, use
Polytetrafluoroethylene (PTFE) disappears to boil tank and disappear to boil to solution to be clarified completely；Disappear after boiling, filtrate is transferred to capacity by the boil liquid that disappears membrane filtration
In bottle, constant volume；
Cd content in measurement soil: taking pedotheque, and after sieving, HNO is added in natural air drying3, HCIO4And HF, disappear and boils to complete
Clarification；The boil liquid that disappears membrane filtration, filtrate is transferred in volumetric flask, constant volume；
With Cd content in atomic absorption spectrometry plant and soil.
10. the verification method that GHH according to claim 7 absorbs the effect of EE2 and Cd to rye grass, which is characterized in that
The specific method of urease activity includes: in measurement soil
Air-dried soil sample is taken, is placed in conical flask, toluene is added and mixes, the urea liquid and citrate buffer added after standing,
Sample is placed in incubator and is sufficiently shaken up, mixture is centrifuged, take supernatant and is diluted with distilled water, by diluted supernatant
Liquid is transferred in centrifuge tube, adds sodium phenate and sodium hypochlorite, and sample is mixed well, and is stood, is diluted with distilled water, use purple
Outside-spectrophotometric measures absorbance, determines NH in sample according to standard curve3The concentration of-N, urease activity U=y × 10 × V ×
W × T, wherein y NH3The concentration of-N；10 be thinner ratio；V is reaction system total volume；W is example weight；When T is reaction
Between；
Measurement Soil Microorganism amount carbon content specific method include:
Moist soil is weighed, is put into conical flask, arginine aqueous solution is added, sample is placed and is cultivated, taking-up is put into refrigerator,
It places, then sample taking-up is placed on and is thawed at room temperature, KCl aqueous solution is added in the completely backward solution that thaws, concussion is extracted, will
Solution is placed in centrifuge tube and is centrifugated, and supernatant membrane filtration measures filtrate；Ammonium content Indophenol blue colorimetry in filtrate
Method measurement；As a result it is calculated as ω (C)=0.01 × NH4 +-Nmg·kg-1, in formula: ω (C) is microbes biomass carbon Bc mass
Score, mgkg-1；0.01 is converted to the coefficient of Bc for arginine induction ammoniation process.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811114767.1A CN109975086A (en) | 2018-09-25 | 2018-09-25 | GHH absorbs effect and the verification method of EE2 and Cd to rye grass |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811114767.1A CN109975086A (en) | 2018-09-25 | 2018-09-25 | GHH absorbs effect and the verification method of EE2 and Cd to rye grass |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109975086A true CN109975086A (en) | 2019-07-05 |
Family
ID=67075982
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811114767.1A Pending CN109975086A (en) | 2018-09-25 | 2018-09-25 | GHH absorbs effect and the verification method of EE2 and Cd to rye grass |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109975086A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110639947A (en) * | 2019-09-27 | 2020-01-03 | 中国计量大学 | Test method for verifying reduction of cadmium absorption of Chinese cabbages |
| CN111052992A (en) * | 2019-12-30 | 2020-04-24 | 中山大学 | Cultivation and management method for improving soil urease activity of field lolium multiflorum |
| CN115655851A (en) * | 2022-10-26 | 2023-01-31 | 安徽省生态环境科学研究院 | A extraction element for restoreing back soil pollution heavy metal concentration determination |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108126976A (en) * | 2018-02-01 | 2018-06-08 | 煜环环境科技有限公司 | A kind of method that chromium-polluted soil is repaired using plant rye grass |
| CN108393338A (en) * | 2017-09-22 | 2018-08-14 | 河南科技大学 | A kind of method and application strengthened rye grass and repair metal strontium polluted soil |
-
2018
- 2018-09-25 CN CN201811114767.1A patent/CN109975086A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108393338A (en) * | 2017-09-22 | 2018-08-14 | 河南科技大学 | A kind of method and application strengthened rye grass and repair metal strontium polluted soil |
| CN108126976A (en) * | 2018-02-01 | 2018-06-08 | 煜环环境科技有限公司 | A kind of method that chromium-polluted soil is repaired using plant rye grass |
Non-Patent Citations (3)
| Title |
|---|
| MARI´A ISABEL ALARCO´N FLORES 等: "QuEChERS-based extraction procedure for multifamily analysis of phytohormones in vegetables by UHPLC-MS/MS", 《J. SEP. SCI.》 * |
| 郭海慧: "EE2降解菌的筛选及其在植物-微生物联合修复污染土壤中的应用", 《中国优秀硕士学位论文全文数据库 工程科技I辑》 * |
| 鲁如坤: "《土壤农业化学分析方法》", 31 December 2000 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110639947A (en) * | 2019-09-27 | 2020-01-03 | 中国计量大学 | Test method for verifying reduction of cadmium absorption of Chinese cabbages |
| CN111052992A (en) * | 2019-12-30 | 2020-04-24 | 中山大学 | Cultivation and management method for improving soil urease activity of field lolium multiflorum |
| CN115655851A (en) * | 2022-10-26 | 2023-01-31 | 安徽省生态环境科学研究院 | A extraction element for restoreing back soil pollution heavy metal concentration determination |
| CN115655851B (en) * | 2022-10-26 | 2023-04-21 | 安徽省生态环境科学研究院 | Extraction device for measuring concentration of heavy metals in soil pollution after remediation |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105950502B (en) | A kind of compound endogenetic bacteria microbial inoculum and its application in heavy-metal contaminated soil phytoremediation | |
| Abid et al. | Biochar increased photosynthetic and accessory pigments in tomato (Solanum lycopersicum L.) plants by reducing cadmium concentration under various irrigation waters | |
| CN107488618B (en) | Bacillus megaterium H3 and application thereof in promoting crop growth and reducing heavy metal absorption of crops | |
| Banuelos et al. | Selenium volatilization in vegetated agricultural drainage sediment from the San Luis Drain, Central California | |
| CN109975086A (en) | GHH absorbs effect and the verification method of EE2 and Cd to rye grass | |
| CN110624949B (en) | Method for repairing excessive phosphorus pollution of phosphorite waste land by combining indigenous microorganisms and plants | |
| Guan et al. | Conversion of Tibetan grasslands to croplands decreases accumulation of microbially synthesized compounds in soil | |
| CN104957009A (en) | Method for regulating turfgrass heavy metal accumulation by using OTC and carbon nano-tube interlayer | |
| CN101337231B (en) | Method for promoting lawn absorb heavy metal from consumer garbage compost and increasing photosynthesis amount cooperated with angleworm | |
| CN110093299A (en) | A kind of salt tolerant growth-promoting bacteria strain D5-2 and its application | |
| CN103232850A (en) | Garbage soil improvement agent and preparation method of improvement agent and improving method of garbage soil | |
| CN104904574A (en) | Method for controlling accumulation of heavy metal in turfgrass by means of siduron and carbon nano tube interlayer | |
| CN101575147B (en) | Method for restoring heavy metal of eutrophic water by utilizing lawn plants | |
| CN114749479B (en) | Method for repairing arsenic-containing gold tailings by utilizing plant-microorganism combination | |
| CN109868242B (en) | Salt-tolerant acetoin-producing bacillus subtilis and application thereof | |
| CN113699059B (en) | Cadmium-resistant growth-promoting paenibacillus strain and application thereof | |
| CN112974505B (en) | Method for restoring rice field soil cadmium pollution by using activator and nitrogen-fixing blue algae | |
| CN110029077A (en) | A kind of salt tolerant growth-promoting bacteria strain Y4 and its application | |
| CN109127712A (en) | A method of utilizing chemistry and microorganism remediation heavy metal pollution tailing | |
| CN109127693A (en) | The method for repairing Cu, Pb, As contaminated soil using sunflower, castor-oil plant inoculation bacillus subtilis | |
| CN109622601B (en) | Method for restoring farmland soil polluted by low-concentration mercury by utilizing cotton and Indian mustard crop rotation | |
| CN103262732B (en) | A kind of method adopting air-dry mud to improve turfgrass plant defence enzyme activity | |
| CN109988738A (en) | A kind of salt tolerant growth-promoting bacteria strain B9 and its application | |
| Larionova et al. | Carbon budget in natural and anthropogenic forest-steppe ecosystems | |
| CN105580688B (en) | A method of honeysuckle Cd accumulation is reduced using Glomus mosseae and improves its yield |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190705 |