CN106011221A - Method for regulating and controlling lawn compost matrix bacterium identification and analysis through carbon nanomaterials - Google Patents
Method for regulating and controlling lawn compost matrix bacterium identification and analysis through carbon nanomaterials Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Abstract
The invention discloses a method for regulating and controlling lawn compost matrix bacterium identification and analysis through carbon nanomaterials. Four kinds of different carbon nanomaterials are mixed in soil to serve as a treatment group, the change of the number of bacteria capable of being cultured in a matrix is studied through lawn plant culture after different carbon nanomaterialss are added for a period of time, and the taxonomic status of microorganisms capable of being cultured in the matrix is analyzed through the molecular sequencing technology. After the carbon nanomaterials are added into a lawn planting system, bacterium class group changes are known, and technical support is provided for application of the carbon nanomaterials to regulating and controlling bacterium class groups in the garbage compost lawn matrix.
Description
Technical field
The invention belongs to environmental protection technical field, relate to a kind of employing carbon nanomaterial regulation and control lawn composting substrate antibacterial
The method of identification and analysis.
Background technology
Carbon nanomaterial is an at least material less than 100 nm in three dimensional structure.Carbon nanomaterial has size
Little, specific surface area is big, and surface can be high, and the feature that surface atom ratio is big shows at aspects such as mechanics, optics, calorifics, electricity
Excellent properties.There is a lot of micro-interface in nano-particle, various interfacial reactions can be strengthened by it, dirty at heavy metal-polluted soil
Dye and sewage purification aspect play obvious action.
Carbon is the highest at nature content, the most extremely important for Organic substance and life entity.1985, scientist
It is found that by 60 former molecular C of C60.CNT and Graphene etc. are found the most in succession subsequently.2004, Geim etc.
People has successfully prepared single-layer graphene.Found successively in the special nature in optical, electrical, thermal and magnetic along with Graphene afterwards,
Research about material with carbon element enters again a brand-new field.
Along with science and technology, being continuously increased of the fast development of modern industry and population, in the world a lot of countries all in
Reveal the trend that water body, soil and atmospheric pollution are the most serious.Report is pointed out, the river of China 50% and the lake of more than 80%
Pool has had been subjected to pollute, and many lakes of III class water standard China of surface water have not reached.The arable land in the whole nation
In have 80,000,000 hm2More than receive pollution in various degree, and distinct issues are exactly organic pollution and weight
The pollution of metal, this situation the most seriously constrains the enforcement of Sustainable Development Strategy of China, has even threatened compatriots'
Healthy.
Although traditional river system repair technology such as cuts dirt, Sediment Dredging etc. temporarily can play some effects, but all controls
Mark does not effects a permanent cure, and biological restoration limiting factor has a lot, such as ambient temperature and Acidity of Aikalinity etc., and this allows for biological restoration visitor
Significant limitation is there is in sight.As traditional contaminated soil remediation method, soil moved in improve the original method, leaching method waste time and energy, and
Well effect is not had for large-area reparation.Compared with traditional environment restoring method, nano material is used for weight
Metallic prosthetic, the absorbability superpower because of it and the specific surface area of super large, the shortcoming that the most there is not traditional method, also show
High remediation efficiency.Additionally atmospheric pollution aspect situation is also SO that is the severeest, that exceed standard in air2, CO and NOxIn the moment
Threaten our healthy.Nano material has splendid catalytic efficiency, even can be catalyzed the most non-incident reaction
New method and thinking is provided for solving generation atmosphere polluting problem.Therefore, utilize nano material solve water body and soil with
And the pollution problem of air is increasingly worth people to pay close attention to.And the research of carbon nanomaterial is the most promising neck
Territory.
Research in recent years finds that CNT can effective heavy metal ion in adsorbed water body.The experiment table of Li etc.
Bright CNT can lead ion in adsorbed water body, and have the biggest dependency with the pH value of solution.Along with solution ph
Increase, the absorbability of lead ion is increased by CNT the most therewith.Langmuir and Freundlich model can solve
Release this adsorption process.Subsequently, Li etc. are investigated the CNT of the different mode of oxidizing process adsorption effect to cadmium ion
Relatively, finding after oxidation processes, the specific surface area of CNT dramatically increases, and the quantity of surface functional group also has substantially increasing
Many.Specifically, CNT is through KMnO4Adsorption effect after oxidation is apparently higher than H2O2And HNO3Oxidation effectiveness.Separately
Outward, next Li et al. has carried out again the experiment of carbon nanotube adsorption copper ion, have studied the CNT of different shape to lead
The absorption of ion is compared, the CNT Adsorption Kinetics to lead ion and the situation of desorption, and discusses carbon
Nanotube is to lead ion, copper ion and cadmium ion competitive Adsorption situation in the same solution.Research finds, CNT pair
It is Pb to the absorption order of three of the above different ions in solution2+＞ Cu2+＞ Cd2+, wherein to lead ion (Pb2+), copper ion
(Cu2+) absorption can with Langmuir equation explain.
Li Yanhui etc. utilize HNO3The CNT of oxidation processes has been carried out Pb2+Adsorption experiment.Research finds HNO3
Carbon nano tube surface after oxidation processes is long-pending to be significantly increased, and additionally the functional group such as-OH ,-CO ,-COOH has been introduced in carbon and receives
The surface of mitron.This can be remarkably reinforced carbon nanotube adsorption Pb2+Active area and active force, thus improve CNT
To Pb2+Adsorbance.Result shows, adsorbance raises along with the rising of temperature, and this explanation CNT is to Pb2+Absorption
It it is an exothermic process.Regeneration test shows, adsorption effect dramatically increases with the increase of pH value.When pH as little as 2, carbon nanometer
The Pb of absorption on pipe2+Desorption rate is to 85%, thus provides theory depend on for realizing recycling of carbon nanotube adsorption material
According to.
In addition, Graphene and graphene oxide for Organic Pollution and are revealed the environmental pollution caused by oil
Also there is the strongest repair.After Graphene modification to the adsorption effect of pollutant in environment more preferably, and adsorbance increase,
Performance is more stable.Chen et al.]The three-dimensional porous graphene oxide film prepared shows huge in terms of selecting absorption
Big potentiality.The weight of its absorption machine oil can reach 37 times of own wt, and the weight of absorbing organic solvent is then more than self
26 times of weight, its adsorption effect compares flake graphite alkene and foamy graphite alkene is much higher.In addition, this porous oxidation
The character of graphene film is sufficiently stable, recycles after the adsorbent on available hexane removal surface.Higher absorption
Amount and longer recycling number of times make this three-dimensional porous graphene oxide film organics removal and cleaning oil pollution
Aspect shows great application prospect.
Above-mentioned analysis shows, the carbon nanomaterial such as Graphene and CNT environmental area application mainly soil,
Water body and atmospheric pollution reparation aspect, and apply as regulator, especially carbon nanomaterial is at compost lawn base
As microorganism adjusting control agent in matter, research there is no document report at present.
Along with the junk produced in China's economic growth and the quickening of urbanization speed, urban life and production process
Present the trend dramatically increased.Meanwhile, the supporting process of house refuse is the most complete, and a large amount of rubbish are exposed to city
In, environment is caused the biggest impact.Composting is urban garbage disposal and resource compares mode easily,
This can not only digest the house refuse in city, alleviates the urban environment pressure that house refuse is brought, and also produces simultaneously
The supply agricultural productions of a large amount of compost fertilizer.
Containing nutrient necessary to a large amount of plant growings in house refuse, therefore consumer garbage compostization is processed also
Compost is used for the effective way that agricultural production is its recycling.
Research finds to add appropriate garbage compost to be effectively increased crop yield in the middle of soil.Because of soil physical chemistry
Matter and agrotype are different, and its amplification there is also significant difference.Zhou Dezhi etc.]After have studied interpolation garbage compost, yellowish soil,
The wheat yield change of plantation in moisture soil and red soil.Result shows, after adding appropriate garbage compost, crop all has volume increase effect
Really, wherein red soil effect is best (crop yield 46%).He Liyuan etc.[43]Research finds, the yield of Plantula Brassicae chinensis is along with rubbish heap
The raising of fertile amount of application and increase, and difference reaches significant level compared with the control.Additionally, the using not only to making produce of compost
Amount has an impact, and also increase significantly effect to the quality of crop.The research of square pavilion et al. finds, adds garbage compost to palm fibre
In red soil, in the seed of soil crop Brassica campestris L, the content of protein significantly raises, and protein contained in soybean kernel is obvious
Increase.
But, people also began to recognize and did not contain only nutrient substance in garbage compost in the last few years, simultaneously possibly together with one
Quantitative heavy metal, if chronic administration can increase the content of heavy metal in soil.Therefore, compost is used for agricultural production may
A series of food-safety problem can be brought.Visible, avoiding food chain, to realize the recycling of garbage compost significant.No
Only such, for preventing compost utilization from causing heavy metal pollution of soil, even bring threat to ecological environment, must while application
The problem that heavy metal is repaired must be considered.
Traditional sod production is typically to utilize high-quality soil.The physical property of soil can be had a huge impact by this,
Fertility is caused to decline.Additionally during sod production, the topsoil soils of 2 more than cm can be pulled away, and fertile topsoil soils enters city
Ecosystem, and after sod production several times, agricultural land soil will become more and more barrenr, so that finally cannot enter again
Row agricultural production, this is waste great to land resources.If turf establishment is using consumer garbage compost as primary structure,
Municipal refuse can not only be digested, additionally it is possible to alleviate turf production and bring the destruction of topsoil soils.
Nanotechnology development in recent years is very rapid, all shows wide application at aspects such as material, information, environment
Prospect.Although there being some to study theoretical research, show that nano material can grow the shadow produced in various degree to microorganism
Ring, but apply its regulating and controlling effect, also there is no correlation technique report.
Summary of the invention
Carbon nanomaterial just has the heavy metal in the biggest range of application, adsorbed water body and soil because of its special structure
It it is the important application of one of which.This technology, from substrate microorganism angle, with consumer garbage compost and soil mixture is
Lawn matrix material, to mix four kinds of different carbon nanomaterials wherein as process group, is cultivated by lawn plant, and research adds
Add after different carbon nanomaterial a period of time cultivable bacteria number change in substrate, and analyze substrate by molecule sequencing technologies
The classification position of middle Bacterial diversity.Recognized by bacterial groups change after carbon nanomaterial is added to turf establishment system
Know, provide technical support for carbon nanomaterial bacterial groups regulation and control application in garbage compost lawn matrix.
The invention discloses following technology contents for achieving the above object:
A kind of method using carbon nanomaterial regulation and control lawn composting substrate Bacteria Identification to analyze, it is characterised in that by following step
Suddenly carry out:
(1) experiment material
Experiment soil takes from Tianjin Normal University's campus 0-20 cm topsoil, and fundamental property is: water content 19.4%, pH
7.27, electrical conductivity 2250 S/cm, phosphorus organic, complete, full nitrogen are respectively 52.3,3.75,2.15 g/kg.
Experiment consumer garbage compost is from Tianjin little Dian destructor plant.PH is 7.49, electrical conductivity 2300 S/cm,
Phosphorus organic, complete, full nitrogen are respectively 132,6.81,25.1 g/kg.Heavy metal Cr, Cu, Pb, Zn, Cd content is respectively 703,
341、217、677、5.01 mg/kg。
Being purchased from Qiu Shi carbon black plant, Tianjin for examination nano carbon black (CB), particle diameter is 20 ~ 70 nm, and specific surface area is 1200 m2/
G, pH value is 7.0, utilizes KMnO before experiment4To its modification.
Graphene (G) microplate is purchased from Nanjing Ji Cang nanosecond science and technology company limited, and structure is black foil shape, microplate size
0.5-20 um, thickness 5-25 nm, specific surface area 40-60 m2/ g, density about 2.25 g/cm3, electrical conductivity 8000-10000 S/
M, phosphorus content > 99.5%.
Graphene oxide (GO) is purchased from Suzhou Heng Qiu nanometer company, for black or brownish yellow powder, average thickness 3.4-7
Nm, lamella diameter 10-50 μm, specific surface area 100-300 m2/ g, purity > 90%.
CNT (CNT) is purchased from Beijing and wins space high-tech new material technology company limited, diameter 20-40 nm, length
10-30 um ,-COOH content 1.43%, purity>90wt%, ashes<8wt%, specific surface area>110 m2/ g, conduction: > 102 s/
cm。
(2) experimental technique:
Measure soil moisture content after fetching earth immediately, be calculated the ratio of soil dry weight and weight in wet base.Proportionally calculate to obtain dry weight
Wet soil weight corresponding to 1500 g.Claim the wet soil of corresponding weight mixes with 30 g garbage composts, stir, loading internal diameter
In the plastic flowerpot of high 20 cm of 15 cm, fill 15 basins altogether.
Experiment sets 5 process, Graphene (G), graphene oxide (GO), CNT (CNT), modified Nano white carbon black altogether
(CB) weigh the ratio of 1% in dry ground to be added separately in mixed-matrix, with the process without carbon nanomaterial for comparison (CK),
Fully mix after adding carbon nanomaterial, every basin sowing tall fescue seed 5g.During cultivation, temperature is 19 ~ 27 DEG C, the wettest
Degree is 60 ~ 72%, and every day keeps the skin wet to soil, makes soil moisture be maintained at the 70% of water capacity.After cultivating 130d, will
In basin, 0-5cm deep soil takes out mixing, crosses 60 mesh sieves, and the preservation of-20 DEG C of refrigerator, for microorganism separation and analysis of accounts.
The present invention is open further has added the method using carbon nanomaterial regulation and control lawn composting substrate Bacteria Identification to analyze
Cultivable bacteria sum in reducing substrate, reduces the application in terms of species diversity index；Total number of bacteria therein refers to
Bacillus cereus (Bacillus), false Xanthomonas campestris (Pseudoxanthomonas), arthrobacterium (Arthrobacter), microbot
Pseudomonas (Microbacterium) and root nodule bacteria (Sinorhizobium).
The present invention also discloses that the method using carbon nanomaterial regulation and control lawn composting substrate Bacteria Identification to analyze exists simultaneously
Improving the application of bacillus cereus quantitative aspects, carbon nanomaterial therein refers to KMnO4To the Nano carbon after its modification
Black.
KMnO is utilized before nano carbon black of the present invention (CB) experiment4Its modification is referred to: weigh nano-sized carbon 10 g
In 250 mL conical flasks, add 100 mL0.03 mol L-1KMnO4Solution, after standing 10 min, is put in general-purpose electric heating
Boiling reflux 1 h on device.After cooling, repeatedly rinse with deionized water, make solution no longer muddy and pH is stable.It is transferred to beaker,
Dry to constant weight under the conditions of 110 DEG C.
More detailed description of the present invention is as follows:
1 develops materials and methods
1.1 experiment material
Experiment soil takes from Tianjin Normal University's campus 0-20 cm topsoil, and fundamental property is: water content 19.4%, pH
7.27, electrical conductivity 2250 S/cm, phosphorus organic, complete, full nitrogen are respectively 52.3,3.75,2.15 g/kg.
Experiment consumer garbage compost is from Tianjin little Dian destructor plant.PH is 7.49, electrical conductivity 2300 S/cm,
Phosphorus organic, complete, full nitrogen are respectively 132,6.81,25.1 g/kg.Heavy metal Cr, Cu, Pb, Zn, Cd content is respectively 703,
341、217、677、5.01 mg/kg。
Being purchased from Qiu Shi carbon black plant, Tianjin for examination nano carbon black (CB), particle diameter is 20 ~ 70 nm, and specific surface area is 1200 m2/
G, pH value is 7.0, utilizes KMnO before experiment4To its modification.
Graphene (G) microplate is purchased from Nanjing Ji Cang nanosecond science and technology company limited, and structure is black foil shape, microplate size
0.5-20 um, thickness 5-25 nm, specific surface area 40-60 m2/ g, density about 2.25 g/cm3, electrical conductivity 8000-10000 S/
M, phosphorus content > 99.5%.
Graphene oxide (GO) is purchased from Suzhou Heng Qiu nanometer company, for black or brownish yellow powder, average thickness 3.4-7
Nm, lamella diameter 10-50 μm, specific surface area 100-300 m2/ g, purity > 90%.
CNT (CNT) is purchased from Beijing and wins space high-tech new material technology company limited, diameter 20-40 nm, length
10-30 um ,-COOH content 1.43%, purity>90wt%, ashes<8wt%, specific surface area>110 m2/ g, conduction: > 102 s/
cm。
Technology design
Measure soil moisture content after fetching earth immediately, be calculated the ratio of soil dry weight and weight in wet base.Proportionally calculate to obtain dry weight
Wet soil weight corresponding to 1500 g.Claim the wet soil of corresponding weight mixes with 30 g garbage composts, stir, loading internal diameter
In the plastic flowerpot of high 20 cm of 15 cm, fill 15 basins altogether.
Experiment sets 5 process, Graphene (G), graphene oxide (GO), CNT (CNT), modified Nano white carbon black altogether
(CB) weigh the ratio of 1% in dry ground to be added separately in mixed-matrix, with the process without carbon nanomaterial for comparison (CK),
Each process repeats for three times.Fully mix after adding carbon nanomaterial, every basin sowing tall fescue seed 5g.During cultivation, temperature is
19 ~ 27 DEG C, relative humidity is 60 ~ 72%, and every day keeps the skin wet to soil, makes soil moisture be maintained at water capacity
70%.After cultivating 130d, 0-5cm deep soil in basin is taken out mixing, cross 60 mesh sieves, the preservation of-20 DEG C of refrigerator, divide for microorganism
From with analysis of accounts.
Method of production
1.3.1 bacteria culture media compound method
According to Microbiology Experiment handbook, carry out the preparation of microbiological culture media.Wherein cultivate antibacterial beef extract-peptone to train
Supporting base (LB culture medium), concrete formula is Carnis Bovis seu Bubali cream 5g, peptone 10g, sodium chloride 5g, agar 18g, distilled water
1000mL, utilizes NaOH or HCl to regulate pH to 7.0～7.2.121 DEG C of sterilizings 20 minutes, when room temperature is dried in the air to 50-60 DEG C, will training
Foster base is poured in the culture dish of diameter 9cm, and 50ml about poured into by each culture dish, fully dries in the air cool to solidification, standby.
The determination of antibacterial culturing optimum concentration
For being smoothed out of testing, before formal experiment, need to first carry out preliminary experiment.According to Microbiology Experiment instruct and
In the past cultivation experience primarily determined that optimum concentration that antibacterial culturing observes was 10-3-10-5Between.Take the substrate in 0.5g matched group
Soil is poured in the conical flask equipped with 50ml sterilized water, and sealing is placed on shaking table, vibrates 20 min with 150 rpm.Vibration
10 S are stood after stopping.Draw l mL soil extract move in test tube and add 9 mL sterilized water, diluted 10 times, system
Become 10-3The soil extract of concentration, utilizes the method to be respectively prepared 10 afterwards-4、10-5The soil extract of concentration.Take 200ul
Culture dish, in LB culture medium, is inverted in the constant incubator of 37 degree by soil extract even spread, and lucifuge is cultivated.Even
Continuous observation bacterial growth situation, counts after its suitable size quantity is stable.Counting and shape for the ease of microorganism
The differentiation of state feature, should choose colony counts culture medium between 30-100 and count, preliminary experiment found that 10-4This
One concentration is the optimum concentration that in the composting substrate of lawn, antibacterial culturing is observed.
Bacterial plate count procedure
Use the antibacterial in the dilution plate rubbing method isolation medium identical with preliminary experiment, and its bacterium colony is counted.Because
Have determined that optimum concentration is 10 by preliminary experiment-4, so lawn composting substrate dilution 10000 times can directly be carried out by experiment
Cultivate.Specific operation process is: weighing 0.5g soil and be poured in the conical flask equipped with 50 mL sterilized water, sealing is placed on
On shaking table, vibrate 20 min with 150 rpm.Vibration stands 10 s after stopping.Draw 0.5 mL soil extract and move into dress
Have in the conical flask of 50ml sterilized water and make 10-4After the soil suspension of concentration, fully mixing, draw 200uL and injected
On solid LB media, coating uniformly, is cultivated 3d in culture dish is inverted in 37 DEG C of constant incubators afterwards, is chosen bacterium colony
Number flat board in the range of 30-300 carries out separating counting, and according to its different colony characteristicses classification, numbering.After sterilizing
Inoculating loop picking bacterial clump culture the most of the same race is inoculated in new LB culture medium the most respectively, draws continuous print broken line,
To reach the purpose to the bacteria purification being separated to.Carry out label after inoculation and be inverted in 37 degree of incubators cultivation 2d, turn afterwards
Move on to 4 DEG C of refrigerators save backup.
The molecular biological variety identification method of antibacterial
Owing to a part of base sequence of the 16SrDNA in bacterial cell is the most conservative, not by environmental condition residing for microorganism
Impact, and a part of sequence is variable, therefore, it can, by the homology of 16S rDNA between com-parison and analysis antibacterial, take off
Show their sibship and Phylogenetic.
Ezup pillar genome DNA extraction test kit (the raw work in Shanghai) is utilized to extract the bacterial genomes that pure culture obtains
DNA, its process requires to carry out according to test kit.Gained genomic DNA is used for the PCR amplification of bacterial 16 S rDNA.
Primer is 27f:
5 '-AGA GTT TGA TCC TGG CTCAG-3 ' 1492r:
5′-GGT TAC CTT GTT ACG ACT T-3′
PCR reaction condition and step: 94 DEG C of denaturations 5min, 94 DEG C of degeneration 1min, 55 DEG C of annealing 45s, 72 DEG C of extension 45s, 30
Individual circulation, 72 DEG C extend 10min.Amplification system is following (50 μ L): primer 1 (27F) 2 μ L；Primer 2 (1492R) 2 μ L；10x
Buffer(Mg2+) 5μL；dNTP 1μL；Taq enzyme 1 μ L；DNA profiling 10 μ L；ddH2O 29μL.Draw different amplified production each
7 L are mixed homogeneously with 2 L 6 × Glycerol DNA loading buffer respectively, draw 7 L mixed liquors and carry out point sample, and
Carry out 1.2% agarose gel electrophoresis, after electrophoresis terminates, agarose gel is immersed dyeing 10min in EB aqueous solution, in ultraviolet
Gel imaging system is observed, it was observed that after bright object band, experiment gained PCR primer is transferred to Beijing Hua Da genome company
Purification and order-checking.
Bacterial 16 S rDNA sequence analysis method
Sequencing result is through NCBI Blast (http: ∥ www.ncbi.nlm.nih.gov) comparison analysis, it is thus achieved that similarity is
The 16SrDNA sequence of five high bacterial strains.ClustalX is used to carry out multi-contrast, carry out phylogeny with MEGA software and divide
Analysis, uses Neighbor-join method phylogenetic tree construction, and number (Bootstrap) of bootstrapping is 1000.Homology journey according to sequence
Degree primarily determines that bacterial strain to be identified status on taxonomy.Cultivation is obtained bacterial strain and the type strain in ncbi database
Compare, if similarity reaches more than 96%, then the bacterial strain that can cultivation be obtained and type strain are classified as a genus, also
Just arrive, by cultivating identification of strains, the level belonged to.
Development results is analyzed
Through cultivation counting and the kind of antibacterial in the composting substrate of lawn are divided and sorted out, pick out the antibacterial of different characteristic
Bacterium colony.
By the PCR to different bacterium 16SrDNA, obtain a plurality of amplified fragments, and by with DNA Marker's
Position versus, finds that its band meets the feature of antibacterial, send the result after survey to show as follows:
This experiment obtains 10 ordered sequences altogether, by the sequence that obtains by Blast program and Gen-Bank data base
Knowing that sequence carries out similarity comparison, its classification information, Similarity Percent and GenBank accession number are shown in Table 1.
The 16SrDNA sequence similarity analysis of cultivable bacteria in table 1 soil
By sequence similarity analysis find bacterial strain 1, bacterial strain 2, bacterial strain 5 and bacterial strain 8 be bacillus (Bacillus), bacterium
Strain 3 be false Xanthomonas (Pseudoxanthomonas), bacterial strain 4 and bacterial strain 10 be Arthrobacter (Arthrobacter),
Bacterial strain 6 and bacterial strain 9 be Microbacterium (Microbacterium), bacterial strain 7 be rhizobium (Sinorhizobium).Similarity
Being all higher than 96%, credibility is higher.
The sequencing result of bacterial isolateses different for isolated 10 strain forms is carried out Blast analysis and multiple ratio pair, it
After utilize MEGA5.1 constructing system cladogram based on 16SrDNA homology.
3 develop conclusion
In conjunction with 16SrDNA, 18SrDNA sequencing technologies, Bacterial diversity in lawn composting substrate after adding carbon nanomaterial is entered
Having gone identification and analysis, main research is as follows: isolated 10 strain antibacterial altogether, shows this 10 strain through 16SrDNA sequencing result
Antibacterial belong to bacillus cereus (Bacillus), false Xanthomonas campestris (Pseudoxanthomonas), arthrobacterium
(Arthrobacter), Microbacterium (Microbacterium) and root nodule bacteria (Sinorhizobium).Add carbon nanomaterial
Can substantially reduce cultivable bacteria sum in substrate, species diversity index also has and declines in various degree.Substrate can be cultivated thin
Bacterium sociales are become bacillus from false xanthomonas, and bacillus advantage is clearly.Nanometer carbon black process group
The quantity of middle bacillus cereus has accounted for the 88.74% of sum.
Accompanying drawing illustrates:
Fig. 1 is that it is carried out the electrophoretogram after 16SrDNA amplification.
Detailed description of the invention
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention
It is method known in those skilled in the art.It addition, embodiment is interpreted as illustrative, and the unrestricted present invention
Scope, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this
On the premise of invention spirit and scope, the various changes carrying out the material component in these embodiments and consumption or change are also
Belong to protection scope of the present invention.The present invention is raw materials used, reagent is commercially available.
Embodiment 1
(1) experiment material
Experiment soil takes from Tianjin Normal University campus 10 cm topsoil, and fundamental property is: water content 19.4%, pH
7.27, electrical conductivity 2250 S/cm, phosphorus organic, complete, full nitrogen are respectively 52.3,3.75,2.15 g/kg.
Experiment consumer garbage compost is from Tianjin little Dian destructor plant.PH is 7.49, electrical conductivity 2300 S/cm,
Phosphorus organic, complete, full nitrogen are respectively 132,6.81,25.1 g/kg.Heavy metal Cr, Cu, Pb, Zn, Cd content is respectively 703,
341、217、677、5.01 mg/kg。
Being purchased from Qiu Shi carbon black plant, Tianjin for examination nano carbon black (CB), particle diameter is 20 nm, and specific surface area is 1200 m2/ g,
PH value is 7.0, utilizes KMnO before experiment4To its modification.
Graphene (G) microplate is purchased from Nanjing Ji Cang nanosecond science and technology company limited, and structure is black foil shape, microplate size
0.5 um, thickness 5 nm, specific surface area 40 m2/ g, density about 2.25 g/cm3, electrical conductivity 8000 S/m, phosphorus content > 99.5%.
Graphene oxide (GO) is purchased from Suzhou Heng Qiu nanometer company, for black or brownish yellow powder, average thickness 3.4-7
Nm, lamella diameter 10 μm, specific surface area 100 m2/ g, purity > 90%.
CNT (CNT) is purchased from Beijing and wins space high-tech new material technology company limited, diameter 20 nm, length
10um ,-COOH content 1.43%, purity>90wt%, ashes<8wt%, specific surface area>110 m2/ g, conduction: > 102 s/cm.
(2) experimental technique:
Measure soil moisture content after fetching earth immediately, be calculated the ratio of soil dry weight and weight in wet base.Proportionally calculate to obtain dry weight
Wet soil weight corresponding to 1500 g.Claim the wet soil of corresponding weight mixes with 30 g garbage composts, stir, loading internal diameter
In the plastic flowerpot of high 20 cm of 15 cm, fill 15 basins altogether.
Experiment sets 5 process, Graphene (G), graphene oxide (GO), CNT (CNT), modified Nano white carbon black altogether
(CB) weigh the ratio of 1% in dry ground to be added separately in mixed-matrix, with the process without carbon nanomaterial for comparison (CK),
Fully mix after adding carbon nanomaterial, every basin sowing tall fescue seed 5g.During cultivation, temperature is 19 DEG C, and relative humidity is
60%, every day keeps the skin wet to soil, makes soil moisture be maintained at the 70% of water capacity.After cultivating 130d, by deep for 1cm in basin
Degree soil takes out mixing, crosses 60 mesh sieves, and the preservation of-20 DEG C of refrigerator, for microorganism separation and analysis of accounts.
Embodiment 2
(1) experiment material
Experiment soil takes from Tianjin Normal University campus 20 cm topsoil, and fundamental property is: water content 19.4%, pH
7.27, electrical conductivity 2250 S/cm, phosphorus organic, complete, full nitrogen are respectively 52.3,3.75,2.15 g/kg.
Experiment consumer garbage compost is from Tianjin little Dian destructor plant.PH is 7.49, electrical conductivity 2300 S/cm,
Phosphorus organic, complete, full nitrogen are respectively 132,6.81,25.1 g/kg.Heavy metal Cr, Cu, Pb, Zn, Cd content is respectively 703,
341、217、677、5.01 mg/kg。
Being purchased from Qiu Shi carbon black plant, Tianjin for examination nano carbon black (CB), particle diameter is 70 nm, and specific surface area is 1200 m2/ g,
PH value is 7.0, utilizes KMnO before experiment4To its modification.
Graphene (G) microplate is purchased from Nanjing Ji Cang nanosecond science and technology company limited, and structure is black foil shape, microplate size 20
Um, thickness 25 nm, specific surface area 60 m2/ g, density about 2.25 g/cm3, electrical conductivity 10000 S/m, phosphorus content > 99.5%.
Graphene oxide (GO) is purchased from Suzhou Heng Qiu nanometer company, for black or brownish yellow powder, average thickness 7 nm, sheet
Layer diameter 50 μm, specific surface area 100-300 m2/ g, purity > 90%.
CNT (CNT) is purchased from Beijing and wins space high-tech new material technology company limited, diameter 40 nm, length 30
Um ,-COOH content 1.43%, purity>90wt%, ashes<8wt%, specific surface area>110 m2/ g, conduction: > 102 s/cm.
(2) experimental technique:
Measure soil moisture content after fetching earth immediately, be calculated the ratio of soil dry weight and weight in wet base.Proportionally calculate to obtain dry weight
Wet soil weight corresponding to 1500 g.Claim the wet soil of corresponding weight mixes with 30 g garbage composts, stir, loading internal diameter
In the plastic flowerpot of high 20 cm of 15 cm, fill 15 basins altogether.
Experiment sets 5 process, Graphene (G), graphene oxide (GO), CNT (CNT), modified Nano white carbon black altogether
(CB) weigh the ratio of 1% in dry ground to be added separately in mixed-matrix, with the process without carbon nanomaterial for comparison (CK),
Fully mix after adding carbon nanomaterial, every basin sowing tall fescue seed 5g.During cultivation, temperature is 27 DEG C, and relative humidity is
72%, every day keeps the skin wet to soil, makes soil moisture be maintained at the 70% of water capacity.After cultivating 130d, by deep for 5cm in basin
Degree soil takes out mixing, crosses 60 mesh sieves, and the preservation of-20 DEG C of refrigerator, for microorganism separation and analysis of accounts.
SEQUENCE LISTING
<110>Tianjin Normal University
<120>method that carbon nanomaterial regulation and control lawn composting substrate Bacteria Identification is analyzed is used
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
agagtttgat cctggctcag 20
<210> 2
<211> 18
<212> DNA
<213>artificial sequence
<400> 2
ggttaccttg ttacgact 18
Claims (4)
1. one kind uses the method that carbon nanomaterial regulation and control lawn composting substrate Bacteria Identification is analyzed, it is characterised in that by following
Step is carried out:
(1) experiment material
Experiment soil takes 0-20 cm topsoil；
Being 20 ~ 70 nm for examination nano carbon black particle diameter, specific surface area is 1200 m2/ g, pH value is 7.0, utilizes KMnO before experiment4
To its modification；
Graphene microchip size 0.5-20 um, thickness 5-25 nm, specific surface area 40-60 m2/ g, density about 2.25 g/cm3, electricity
Conductance 8000-10000 S/m, phosphorus content > 99.5%；
Graphene oxide average thickness 3.4-7 nm, lamella diameter 10-50 μm, specific surface area 100-300 m2/ g, purity > 90%；
Carbon nanotube diameter 20-40 nm, length 10-30 um ,-COOH content 1.43%, purity>90wt%, ashes<8wt%, than
Surface area > 110 m2/ g, conduction: > 102 s/cm；
(2) experimental technique:
Measure soil moisture content after fetching earth immediately, be calculated the ratio of soil dry weight and weight in wet base, proportionally calculate to obtain dry weight
Wet soil weight corresponding to 1500 g, claim the wet soil of corresponding weight mixes with 30 g garbage composts, stir, loading internal diameter
In the plastic flowerpot of high 20 cm of 15 cm, fill 15 basins altogether；
Experiment sets 5 process altogether: Graphene (G), graphene oxide (GO), CNT (CNT), modified Nano white carbon black (CB) are pressed
Dry ground weighs the ratio of 1% and is separately added into, and fully mixes, be right with the process without carbon nanomaterial after adding carbon nanomaterial
According to (CK), every basin sowing tall fescue seed 5g, during cultivation, temperature is 19 ~ 27 DEG C, and relative humidity is 60 ~ 72%, and every day is given
Soil keeps the skin wet, and makes soil moisture be maintained at the 70% of water capacity, after cultivating 130d, is taken by 0-5cm deep soil in basin
Going out mixing, cross 60 mesh sieves, the preservation of-20 DEG C of refrigerator, for microorganism separation and analysis of accounts.
2. the method described in claim 1, wherein the fundamental property of soil is: water content 19.4%, pH 7.27, electrical conductivity 2250
S/cm, phosphorus organic, complete, full nitrogen are respectively 52.3,3.75,2.15 g/kg；
Experiment consumer garbage compost is from Tianjin little Dian destructor plant, and pH is 7.49, and electrical conductivity 2300 S/cm is organic
Matter, full phosphorus, full nitrogen are respectively 132,6.81,25.1 g/kg；
Heavy metal Cr, Cu, Pb, Zn, Cd content are respectively 703,341,217,677,5.01 mg/kg；
Nano carbon black, particle diameter is 20 ~ 70 nm, and specific surface area is 1200 m2/ g, pH value is 7.0, utilizes KMnO before experiment4Right
Its modification；
Graphene microchip size 0.5-20 um, thickness 5-25 nm, specific surface area 40-60 m2/ g, density about 2.25 g/cm3, electricity
Conductance 8000-10000 S/m, phosphorus content > 99.5%；
Graphene oxide average thickness 3.4-7 nm, lamella diameter 10-50 μm, specific surface area 100-300 m2/ g, purity > 90%；
CNT, diameter 20-40 nm, length 10-30 um ,-COOH content 1.43%, purity>90wt%, ashes<8wt%, table
Area > 110 m2/ g, conduction: > 102 s/cm.
3. the method using carbon nanomaterial regulation and control lawn composting substrate Bacteria Identification to analyze described in claim 1 is reducing substrate
Middle cultivable bacteria sum, reduces the application in terms of species diversity index；Total number of bacteria therein refers to bacillus cereus
(Bacillus), false Xanthomonas campestris (Pseudoxanthomonas), arthrobacterium (Arthrobacter), Microbacterium
(Microbacterium) and root nodule bacteria (Sinorhizobium).
4. the method using carbon nanomaterial regulation and control lawn composting substrate Bacteria Identification to analyze described in claim 1 is improving spore
The application of bacillus quantitative aspects, carbon nanomaterial therein refers to KMnO4To the nano carbon black after its modification.
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