CN108977431A - Bagasse immobilization microorganism Aspergillus aculeatus and its application in quinolone antibiotics pollution amelioration - Google Patents
Bagasse immobilization microorganism Aspergillus aculeatus and its application in quinolone antibiotics pollution amelioration Download PDFInfo
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- CN108977431A CN108977431A CN201810659294.7A CN201810659294A CN108977431A CN 108977431 A CN108977431 A CN 108977431A CN 201810659294 A CN201810659294 A CN 201810659294A CN 108977431 A CN108977431 A CN 108977431A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
- C12N11/12—Cellulose or derivatives thereof
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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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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/347—Use of yeasts or fungi
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C2101/00—In situ
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/38—Organic compounds containing nitrogen
Abstract
The invention discloses bagasse immobilization microorganism Aspergillus aculeatus and its applications in quinolone antibiotics pollution amelioration, use bagasse for the fixed degradation bacteria microorganism Aspergillus aculeatus of fixation support, bagasse is pre-dried, it is pre-processed before inoculation using inorganic salts culture solution, bacteria suspension adds the mixed liquor of inorganic salts culture solution and the amount ratio of bagasse is 10mL:1g, and microorganism Aspergillus aculeatus strains A spergillus-438 bacteria suspension and the amount ratio for drying bagasse are 0.2~5mL ︰ 1g.Bagasse immobilization microorganism Aspergillus aculeatus can repair in water and quinolone antibiotics pollution in soil.The present invention is at low cost; have the characteristics that bacterial density is big, reaction speed is fast and resistance to environmental impact; and it is cheap environmentally friendly, without secondary pollution as carrier; there is in quinolone antibiotics pollution amelioration very big application potential in water body and soil, to preserve the ecological environment, agricultural production, human health have positive effect.
Description
[technical field]
The present invention relates to bagasse immobilization microorganism Aspergillus aculeatus and its applications in quinolone antibiotics pollution amelioration, especially
It is the Ciprofloxacin in water body and soil, the application in Norfloxacin pollution amelioration, belongs to immobilized microorganism restoration of the ecosystem
Technical field.
[background technique]
Quinolone antibiotics are a kind of artificial synthesized general antibacterial agents of people and animals, belong to pyrrone acid derivative.There is antibacterial
The features such as spectrum is wide, antibacterial activity is strong and other antibacterials are without cross resistance, has a better effect multiple drug-resistant bacteria, often
It is used as the treatment infection of penicillin and cephalosporin alternative medicine, has in treatment humans and animals bacterial infection disease good
Effect is widely used in the treatment and prevention of Animal diseases, is added in animal feed frequently as animal and fowl fodder addition drug.
At present both at home and abroad using it is more be third generation quinolone, also known as fluoquinolone Fluorquinolones (FQs), representative has
(ENR) such as Ciprofloxacin (CIP), Norfloxacin (NOR), lavo-ofloxacin (LEV), Enrofloxacins, gatifloxacin (GAT).
Investigation display, antibiotic total usage amount in China's is about 16.2 ten thousand tons within 2013, and wherein fluoquinolone usage amount is
25500 tons, usage amount is larger, and is in growing trend.Metabolic rate is lower in animal body for quinolone antibiotics, mostly with original
Medicine or other metabolisable forms are exhausted directly in vitro with excrement, urine, and the excreta of livestock and poultry is that environment quinolones is anti-
One of main source of raw element pollution, the quinolone antibiotics bioactivity still with higher after discharge.There is research
20, Guangdong Province scale pigs and cattle farm excrement is tested and analyzed, 4 kinds of quinolone antibiotics and 4 kinds of sulfamido antibiosis
Element is detected, the former fresh weight total content is between 24.5~1516.2 μ g/kg, 581.0 μ g/kg of average out to, with Ciprofloxacin
Based on Enrofloxacin.Solubility is lower in water for quinolone antibiotics, and easily with Ca in soil2+、Mg2+Equal metals sun from
Son is combined into stable comple, is easy to be retained by adsorption by soil, and quinolone antibiotics mainly returns to become for soil in environment.
There is research for statistical analysis to the content of the fluoroquinolone antibiotics in the pedotheque of Southeastern Suburb of Beijing 86, finds soil
The average value of 5 kinds of fluoroquinolone antibiotics total contents is 46.1 μ g/kg in sample.In the case where being protected from light natural conditions, fluoquinolone
Class antibiotic is highly stable, not degradable, and wherein the half-life period of Ciprofloxacin is 462.1d at 1 year or more, for persistently pollution
Object.Untreated feces of livestock and poultry is directly applied to farmland as organic fertilizer, has persistent quinolone antibiotics through rain
Water elution enters in surface water and groundwater, constantly accumulates in soil, causes the pollution aggravation of soil quinolone antibiotics, right
The stabilization of ecological environment and the health of organism threaten.Quinolone antibiotics in soil can by it is plants enriched, through eating
The transmitting of object chain.In recent years, quinolone antibiotics not only detect in soil, water body, and the report detected in vegetables, meat egg is also
It is more and more.There is research to carry out detection and analysis discovery, the city Zhu Sanjiaoduo to the antibiotic in the Vegetable Base soil of Pearl River Delta area
Quinolone antibiotics mean concentration is 122.25 μ g/kg in the organic vegetable base of detection.It is come from from the acquisition of Guangzhou supermarket
Detect discovery in 41 parts of various vegetable samples of the ground such as Guangdong, Shandong, the recall rates of quinolone antibiotics is 93% or more, promise
The highest content of Flucloxacillin, Ciprofloxacin and Enrofloxacin is respectively 148.92,108.11 and 85.19 μ g/kg.As Novel ring
Border pollutant, pollution remediation technology have received widespread attention.
Quinolone antibiotics pollution can be handled by the method for physics, chemistry or biology.Physical method project amount
Greatly, removal effect is limited, including physisorphtion, membrane processing method etc.；When removing hardly degraded organic substance, chemical method is shown well
Effect, chemical method include photocatalytic degradation method, hydrolysis, electrochemical oxidation process etc., but the reactant or chemistry of chemical method addition
The lower intermediate product generated of effect may have stronger toxicity, more difficult to degrade；Biodegrade is remove environmental contaminants one
Kind effective mode, including activated-sludge method, activation process etc..Suitable microorganism can utilize target contaminant, pass through one
To make object be metabolized as carbon dioxide and water etc., the method is safe, at low cost and without secondary pollution, has caused the country for series effect
The extensive concern of outer scholar.
Existing method is more the reparation for paying close attention to quinolone antibiotics polluted-water, to quinolone antibiotics in soil
It studies less or bad to soil quinolone antibiotics treatment effect.The handled antibiotic concentration of many researchs is lower, with
The concentrations gap of quinolone antibiotics is larger in recent years.
Immobilized microorganism technique is primarily referred to as being fixed on the free microorganism of dispersion by method physically or chemically
The area of space of restriction, to improve the concentration of microbial cell, the method for making it keep higher bioactivity and recycling.
The advantageous microenvironment that carrier material can provide as buffer system, shield indigenous microorganism Competition and unfavorable external condition
Infringement, thus guarantee inoculation efficient degradation microorganism good growth；Fixation support can be with as adsorbent simultaneously
The quinolone antibiotics being effectively enriched in water body or soil, improve its biologically effective concentration on carrier；Microorganism and
Its ectoenzyme secreted also is fixed on carrier by enrichment, increases the contacting efficiency with extracellular antibiotic, realizes efficient degradation
Bacterium effectively repairs antibiotic pollution.
It is mostly the not high organic carrier of higher cost, bioaffinity currently used as microbial immobilized carrier, such as penta
Dialdehyde, calcium alginate, sodium alginate, polyvinyl alcohol and polyacrylamide gel etc..Agricultural wastes (such as bagasse, peanut shell and
Stalk etc.) as microbial immobilized carrier, there is very strong compatibility to the microorganism being grown in its surface and hole；Also
Quinolone antibiotics pollutant in adsorbable Enriching soil improves the efficient degradation microorganism for being fixed on its surface and hole
The efficiency for quinolone antibiotics of degrading；Agricultural wastes nutrient rich in, in use process can with natural degradation,
Organic mass-energy after degradation improves soil physico-chemical property and not will cause secondary pollution, and valence so the processing after is simple and environmentally-friendly
Lattice are cheap.
[summary of the invention]
The invention aims to overcome the deficiencies of the prior art and provide a kind of to handle quinolones in water body and soil
The bagasse immobilization microorganism Aspergillus aculeatus of antibiotic pollution, and provide that a kind of strain density is big, reaction speed is fast, the fixation of resistance to environmental impact
Change high residue, quinolone antibiotics Ciprofloxacin difficult to degrade, Norfloxacin in microbial process processing water body and soil, fits
It is polluted for repairing quinolone antibiotics in water body and soil.
The purpose of the present invention is what is be achieved through the following technical solutions:
Bagasse immobilization microorganism Aspergillus aculeatus, it is characterised in that use bagasse for the fixed degradation bacteria microorganism Aspergillus aculeatus of fixation support
Aspergillus-438。
Bagasse in the present invention is pre-dried, and pre-processes before inoculation using inorganic salts culture solution, and bacteria suspension adds inorganic salts
The mixed liquor of culture solution and the amount ratio of bagasse are 10mL:1g.
Microorganism Aspergillus aculeatus strains A spergillus-438 in the present invention first passes through culture in advance before being immobilized onto bagasse carrier
It is prepared into bacteria suspension, the bacteria suspension and the amount ratio for drying bagasse are 0.2~5mL ︰ 1g.
Microorganism Aspergillus aculeatus Aspergillus-438 bacteria suspension in the present invention is prepared by following steps culture:
By microbionation after purification on the solid screening and culturing medium in test tube, 28 DEG C are activated 2 days, by the bacterium in test tube
Strain normal saline flushing is simultaneously inoculated in the conical flask that volume is 50mL liquid screening medium, in 28 DEG C, 150r/min's
It is spare to be placed in 4 DEG C of refrigerators to logarithmic growth phase for vortex oscillation culture on shaking table.
In the present invention on bagasse carrier the step of immobilization microorganism Aspergillus aculeatus Aspergillus-438 are as follows:
A, bagasse cleaned into drying, smashed, conical flask is added, high pressure steam sterilization handles 30min, cooling；
B, the inorganic salts culture solution of aseptic process, ultraviolet sterilization 40min are added to conical flask；
C, inoculated and cultured mixes sealing, in 28 to the microorganism Aspergillus aculeatus Aspergillus-438 bacteria suspension of logarithmic growth phase
DEG C, revolving speed is for shaken cultivation in the shaking table of 150r/min to microbes biomass maximum up to bagasse immobilization microorganism Aspergillus aculeatus
Aspergillus-438。
A kind of application of bagasse immobilization microorganism Aspergillus aculeatus in quinolone antibiotics pollution amelioration.
Bagasse immobilization microorganism Aspergillus aculeatus Aspergillus-438 of the present invention quinolone antibiotics Ciprofloxacin in water
In pollution amelioration, the dosage of bagasse immobilization bacterium are as follows: dry bagasse carrier and microorganism Aspergillus aculeatus Aspergillus-438 bacteria suspension
Amount ratio be 1g:2.5-4.5mL, preferably 1g:3.5mL, the Ciprofloxacin contaminant water of 40 times of quality of bagasse can be administered.When processing
It is mixed according to the ratio, concussion is handled at 32-43 DEG C, 150r/min.
Bagasse immobilization microorganism Aspergillus aculeatus Aspergillus-438 of the present invention quinolone antibiotics Ciprofloxacin in the soil
Pollution amelioration in, the dosage of bagasse immobilization bacterium are as follows: dry bagasse carrier and microorganism Aspergillus aculeatus Aspergillus-438 bacterium are outstanding
The ratio of liquid is 1g:0.2-2mL, preferably 1g:1mL, can handle the Ciprofloxacin contaminated soil of 10 times of weight of dry bagasse.Processing
When be uniformly mixed according to the ratio, cultivated at 28 DEG C~43 DEG C, soil kept to have certain humidity, water content 40%-60%,
To guarantee bacterial growth.
Bagasse immobilization microorganism Aspergillus aculeatus Aspergillus-438 of the present invention quinolone antibiotics Norfloxacin in water
In pollution amelioration, the dosage of bagasse immobilization bacterium are as follows: dry bagasse carrier and microorganism Aspergillus aculeatus Aspergillus-438 bacteria suspension
Amount ratio be 1g:3-5mL, preferably 1g:4mL, the Norfloxacin contaminant water of 40 times of quality of bagasse can be administered.According to this when processing
Ratio mixing, concussion is handled at 37 DEG C, 150r/min.
Bagasse immobilization microorganism Aspergillus aculeatus Aspergillus-438 of the present invention quinolone antibiotics Norfloxacin in the soil
Pollution amelioration in, the dosage of bagasse immobilization bacterium are as follows: dry bagasse carrier 1g is inoculated with 0.5-1.5mL microorganism Aspergillus aculeatus
Aspergillus-438 bacteria suspension, preferably dry carrier 1g, is inoculated with 1mL microorganism Aspergillus aculeatus Aspergillus-438 bacteria suspension, can
Handle the Ciprofloxacin contaminated soil of dry 10 times of weight of bagasse.It is uniformly mixed when processing according to the ratio, at 28 DEG C~43 DEG C
Lower culture keeps soil to have certain humidity, water content 40%-60%, to guarantee bacterial growth.
Compared with prior art, the present invention has the following advantages:
Fixation support uses biological material bagasse, and bagasse is that agricultural residue is at low cost, obtains and is easy, former for environment
Position repair when can natural degradation, environmental protection it is without secondary pollution；Immobilization is easy to operate, and immobilized microorganism is easy to use, can be a large amount of
Preparation is promoted；The pollution of Ciprofloxacin can reach preferable treatment effect in processing water body and soil, which can reduce
The risk that quinolone antibiotics are enriched in the environment avoids quinolone antibiotics pollution from transmitting by food chain, to ecology
Environment and human health are of great significance to.
The present invention is screened by defined medium, tames out the spy for having efficient degradation to act on Ciprofloxacin, Norfloxacin
Determine degradation bacteria microorganism Aspergillus aculeatus Aspergillus-438, and use bagasse for fixation support, immobilization constructs efficient stable
Agricultural residue immobilized microorganism system.This method immobilized microorganism big, fast and resistance to environment of reaction speed with bacterial density
The features such as impact, and as the cheap environmental protection of carrier, without secondary pollution, the quinolone antibiotics pollution amelioration in water body and soil
In there is very big application potential, to preserve the ecological environment, agricultural production, human health have positive effect.
[Detailed description of the invention]
Fig. 1 is the scanning electron microscope (SEM) photograph of the microorganism Aspergillus aculeatus Aspergillus-438 in the present invention；
Fig. 2 is the microbial growth curve figure of the microorganism Aspergillus aculeatus Aspergillus-438 bacteria suspension in the present invention
(OD600nm)；
Fig. 3 is the degradation effect figure of bagasse immobilization Aspergillus-438 of the present invention Ciprofloxacin when handling sewage；
Fig. 4 is the influence that different temperatures handles bagasse immobilization Aspergillus-438 Ciprofloxacin contaminated soil；
Fig. 5 is the influence that heavy metal ion handles bagasse immobilization Aspergillus-438 Ciprofloxacin contaminated soil；
Fig. 6 is the influence that difference connects that bacterium amount handles bagasse immobilization Aspergillus-438 Norfloxacin contaminated soil.
[specific embodiment]
The method of bagasse immobilization microorganism Aspergillus aculeatus of the present invention includes the following steps:
1. by enrichment culture, isolating and purifying and taming the spine spore that obtains and can be grown using Ciprofloxacin as sole carbon source
Aspergillus Aspergillus-438 bacterium；
2. microorganism Aspergillus aculeatus Aspergillus-438 bacterium after purification to be inoculated in the inclined-plane of solid screening and culturing medium in test tube
Reach saturation to bacterium colony growth within upper activation two days, by the strain inoculated on inclined-plane in 50mL liquid screening medium, is shaken in 28 DEG C
It swings in incubator and two days obtained bacteria suspensions of culture is shaken up with 150r/min, it is spare in 4 DEG C of refrigerators；
3. by the dry bagasse smashed weigh it is appropriate conical flask is added, high pressure steam sterilization processing is cooling, to conical flask plus
Enter the inorganic salts culture solution of aseptic process, then ultraviolet sterilization is inoculated with microorganism Aspergillus aculeatus Aspergillus-438 bacteria suspension, mix
Sealing, in 28 DEG C, revolving speed is that shaken cultivation is maximum to microbes biomass in the shaking table of 150r/min；
Wherein bacteria suspension adds the mixed liquor of inorganic salts culture solution and the amount ratio of bagasse to be 10mL:1g, bacteria suspension and drying
The amount ratio of bagasse is 0.2~5mL ︰ 1g, and inorganic salts culture solution is supplied.
The present invention is described in further detail combined with specific embodiments below:
The step of bagasse immobilization microorganism Aspergillus aculeatus, is as follows:
I, screening, separation, the domestication of degradation bacteria microorganism Aspergillus aculeatus Aspergillus-438
Culture medium needed for testing is as follows:
Trace element solution (1000mL): FeSO40.1g, MnSO40.1g, ZnSO40.1g, Na2MoO40.01g,
CaCl20.1g, CuSO40.1g, distilled water 1000mL.
Inorganic salts culture solution (1000mL): K2HPO40.5g, KH2PO40.5g, NaCl 0.2g, MgSO40.2g,
NH4NO31.0g, trace element solution 10mL, distilled water l000mL, pH=7.5；
Liquid screening medium (1000mL): K2HPO40.5g, KH2PO40.5g, NaCl0.2g, MgSO40.2g,
NH4NO31.0g, trace element solution 10mL, distilled water l000mL, Ciprofloxacin are added by simulating pollution object demand, pH=
7.5；Ciprofloxacin is the inorganic salts culture solution of sole carbon source
Solid screening and culturing medium need to add 20~25g agar in liquid screening medium；
It is derived from fresh pig manure, scale livestock farming sampling each 5g of soil on Guangzhou pig farm, sterile water 95mL is placed in
In 250mL conical flask, appropriate bead is added, in vibrating 20min on 28 DEG C, the shaking table of 150r/min, obtains containing bacterium stoste.It takes
The 1mL stoste is uniformly coated on spreading rod using Ciprofloxacin as sole carbon source, and the solid that pollution concentration is 50mg/L screens training
It supports on base；It is cultivated 3 days under the conditions of 28 DEG C, bacterium colony growing state on culture medium is observed in timing.Carefully choose bacterium colony on culture medium, adopts
With plate streak to pick out thallus separates, the single bacterium colony purified.Using Ciprofloxacin as sole carbon source, press
It successively transfers according to the solid screening and culturing medium that pollution concentration is 50mg/L, 100mg/L, 150mg/L, 200mg/L, 250mg/L, often
A concentration is transferred 3 times, and the tolerance degradation bacteria of domestication is finally obtained.Under the purifying bacterium isolated is rinsed respectively with physiological saline
Come, is seeded on the liquid screening medium that pollution concentration is 50mg/L, is vibrated on 28 DEG C, the shaking table of 150r/min, with height
Effect liquid phase chromatogram monitors remaining Ciprofloxacin content in 24 hours, detects the degradation effect of Ciprofloxacin, chooses degradation efficiency
Highest bacterial strain is forwarded on the inclined-plane solid screening and culturing medium that Ciprofloxacin Concentration is 250mg/L, and 4 DEG C save backup,
Carry out subsequent experimental.
Resulting degradation bacteria microorganism Aspergillus aculeatus Aspergillus-438 is cultivated 3 days on solid screening and culturing medium, observes its bacterium
Fall form: being black particle after mature to be light yellow to khaki when initial, quality be slightly in it is cotton-shaped, closely intersperse among entire flat
In plate.It is oval irregular shape that (as shown in Figure 1) visible Aspergillus-438 is observed under scanning electron microscope, and wall surface is not only
It is sliding, there is more short thorn.Its biological characteristics are as follows: catalase experiment is positive, and is aerobic power bacterium；Methyl red experiment is in sun
Property；Indoles experiment is produced to be positive；Starch Hydrolysis experiment is positive.The physio-biochemical characteristics of bacterial strain are according to " clinical common bacteria is true
Dientification of bacteria handbook " it carries out.By known strain sequence in the nucleotide sequence qualification result of Aspergillus-438 and ncbi database
Column carry out homologous similitude comparison, and sequence is reached with microorganism Aspergillus aculeatus category (Aspergillusaculeatus) homologous similarity
100%.
II, microorganism Aspergillus aculeatus Aspergillus-438 bacteria suspension is prepared
The poly- I of the step microorganism Aspergillus aculeatus Aspergillus-438 bacterium obtained is inoculated in the oblique of solid screening and culturing medium in test tube
It activates 2 days on face, by the strain inoculated on inclined-plane in 50mL liquid screening medium, is vortexed on 28 DEG C, the shaking table of 150r/min
Shaken cultivation is to logarithmic growth phase, and taking around 60~64 hours, (growth curve of microorganism Aspergillus aculeatus Aspergillus-438 is as schemed
Shown in 2), take supernatant to pour out after centrifugation, bacteria suspension is made, and to be placed in 4 DEG C of refrigerators spare.
III, bagasse immobilization degradation bacteria microorganism Aspergillus aculeatus Aspergillus-438
Bagasse cleans drying, is cut into a section shape, is moderately smashed with pulverizer, weighs appropriate addition conical flask, 1.5Mpa high pressure
Steam sterilizing handles 30min, and to conical flask plus the inorganic salts culture solution of aseptic process, sterilize 40min under ultraviolet sterilization ultraviolet lamp,
To be cooled, bacteria suspension adds the mixed liquor of inorganic salts culture solution and the amount ratio of bagasse to be 10mL:1g；It is connect on processed bagasse
Kind of culture mixes sealing, in 28 DEG C, revolving speed 150r/ to the microorganism Aspergillus aculeatus Aspergillus-438 bacteria suspension of logarithmic growth phase
Shaken cultivation is to microbes biomass maximum up to bagasse immobilization microorganism Aspergillus aculeatus Aspergillus-438 in the shaking table of min；It reaches
To microbes biomass maximum demand culture 2~3 days.The amount ratio of bacteria suspension and dry bagasse is 0.2~5mL ︰ 1g, is applied to
The preferred bacteria suspension of processing of Norfloxacin and the amount ratio of dry bagasse are 1mL ︰ 1g in soil, are applied to Ciprofloxacin in sewage
The preferred bacteria suspension of processing and the amount ratio of dry bagasse be 3.5mL ︰ 1g.
Application examples 1: bagasse immobilization microorganism Aspergillus aculeatus Aspergillus-438 handles Ciprofloxacin sewage
3 kinds of experimental setup processing: (immobilization Aspergillus-438+ Ciprofloxacin is dirty for immobilized microorganism experimental group
Water)；Free microorganism control group (Aspergillus-438 bacteria suspension+Ciprofloxacin sewage)；Blank control group (Ciprofloxacin
Sewage).
Amount ratio by bacteria suspension and dry bagasse is that 3.5mL ︰ 1g prepares 3 groups of bagasse immobilization Aspergillus-438,
Free microorganism and blank control group control experiment with parallel culture.
Preparation simulation Ciprofloxacin sewage, it is husky that each conical flask is separately added into the cyclopropyl that 100mL pollution concentration is 100mg/L
Star liquid screening medium, sealing are wrapped.Shaken cultivation 96 hours, use high performance liquid chromatography in 28 DEG C, 150r/min shaking table
Sample detection.As a result fig. 3, it is shown that blank control group and free control group are without obvious degradation, 12h in 96 hours
Interior bagasse immobilization microorganism Aspergillus aculeatus Aspergillus-438, up to 51.74%, illustrates to fix to the Ciprofloxacin removal rate in sewage
Change microorganism and free microorganism is substantially better than to the removal effect of Ciprofloxacin sewage.
Application examples 2: bagasse immobilization microorganism Aspergillus aculeatus Aspergillus-438 handles Ciprofloxacin contaminated soil
2.1 tests are derived from Agricultural University Of South China's arboretum with soil, and quality is silt red earth, soil pH (water/soil=
It 2/1) is 5.25, organic matter 7.89g/kg, nitrogen, phosphorus, potassium are other 0.71,0.72,2.61g/kg.Ciprofloxacin, mistake is not detected
It is stand-by after 2mm sieve.100mg Ciprofloxacin is dissolved with weak aqua ammonia, adds in 50g soil, is uniformly mixed, after room-dry
The manual simulation's soil for being 100mg/kg to get pollution concentration is uniformly mixed with 950g soil again, camera bellows saves stand-by.
Influence of 2.2 different temperatures to bagasse immobilization Aspergillus-438 processing Ciprofloxacin contaminated soil.
The following 3 kinds of processing of test setting: blank control group (the Ciprofloxacin Polluted Soil that bacterium is not added)；Free microorganism control
Group (Aspergillus-438 bacteria suspension+Ciprofloxacin Polluted Soil)；Under 5 kinds of different temperatures immobilized microorganism experimental group (Gu
Surely change Aspergillus-438+ Ciprofloxacin Polluted Soil).0d rises, and every 5d extracting sampling observes 30d, every kind of processing sets 3 altogether
In parallel.
Amount ratio by bacteria suspension and dry bagasse is that 3.5mL ︰ 1g prepares 3 groups of bagasse immobilization Aspergillus-438,
Free microorganism and blank control group control experiment with parallel culture.
The Ciprofloxacin Polluted Soil of 10g is added into beaker, stirs evenly, by immobilization microorganism Aspergillus aculeatus Aspergillus-
438 experimental group groups be respectively put into 23 DEG C, 28 DEG C, 33 DEG C, 38 DEG C, cultivate in 43 DEG C of biochemical cultivation case, free microorganism and sky
White control group is placed in 28 DEG C of cultures, keeps soil to have certain moisture in experiment to guarantee that microorganism grows.Respectively at the 0th, 5,
10,15,20,25 and 30d sampling extracting, with the residual quantity of high effective liquid chromatography for measuring Ciprofloxacin and calculates its degradation rate.
From fig. 4, it can be seen that the degradation rate of 23 DEG C, 28 DEG C, 33 DEG C, 38 DEG C, 43 DEG C Ciprofloxacins divides in immobilized microorganism group
It Wei 28.32%, 35.46%, 41.39%, 46.03%, 58.24%.Compared in blank control processing, free microorganism processing
The degradation rate of Ciprofloxacin is respectively as follows: 2.89%, 1.94%, and the effect of immobilization bacterium processing Ciprofloxacin soil is substantially better than
Free microorganism processing；Degradation effect is increased with temperature and is increased in immobilized microorganism group, and possible cause reaches enzyme activity
Suitable temperature, but temperature it is excessively high may to microorganism grow it is unfavorable.The bacterium southern area damp and hot for warmer climate is very suitable
With.
Influence of 2.3 heavy metal ion to bagasse immobilization Aspergillus-438 processing Ciprofloxacin contaminated soil.
Heavy metal ion pollution in soil is got worse, and heavy metal ion Cd is chosen in experiment2+And Pb2+Probe into heavy metal
Bagasse immobilization Aspergillus-438 handles the effect of Ciprofloxacin contaminated soil in the presence of ion.In 100mg/kg cyclopropyl
On the basis of husky star Polluted Soil, according to soil heavy metal ion pollution situation, heavy metal ion Cd is prepared respectively2+Pollution concentration
For 10mg/kg, heavy metal ion Pb2+Pollution concentration be 100mg/kg simulation soil.3 groups of controls of experimental setup, using solid
Surely change control group soil, heavy metal ion Cd that Aspergillus-438 processing is free of heavy metal ion2+Contaminated soil and again
Metal ion Pb2+Contaminated soil.The total content of bacteria suspension and inorganic salts culture solution is 10mL, handles Ciprofloxacin contaminated soil
10g is placed in 28 DEG C of cultures, residual with high effective liquid chromatography for measuring Ciprofloxacin respectively at the 0th, 5,10,15d sampling extracting
Allowance simultaneously calculates its degradation rate.
As seen from Figure 5, if other conditions are the same, after cultivating 15d, bagasse immobilization Aspergillus-438
To Cd containing heavy metal ion2+Contaminated soil in Ciprofloxacin degradation rate be 48.69%, to Pb containing heavy metal ion2+Dirt
Ciprofloxacin degradation rate in dye soil is 40.75%, is to the control group soil degrading rate without heavy metal ion
62.23%.It can be seen that heavy metal ion pollution has certain inhibiting effect, heavy metal ion Pb for the growth of microorganism2+Suppression
Production is with stronger, but bagasse immobilization Aspergillus-438 remains to make Ciprofloxacin degradation in soil, illustrates bagasse immobilization
Aspergillus-438 sugarcane not only has the ability of Ciprofloxacin in stronger degradation soil, but also has certain environment to adapt to
Ability.
Application examples 3: bagasse immobilization microorganism Aspergillus aculeatus Aspergillus-438 handles Norfloxacin contaminated soil
3.1 Norfloxacin contaminated soils, which are pressed, applies method preparation, difference in 2 to connect bacterium amount to bagasse immobilization
The influence of Aspergillus-438 processing Norfloxacin contaminated soil, it may be assumed that trained by after different gradients addition bacteria suspension with inorganic salts
Nutrient solution is supplemented to 10mL, makes the amount ratio 10mL ︰ 1g of mixed liquor and dry bagasse
3.2 in immobilized microorganism experimental group, the amount of bacteria suspension used in immobilization is 1%, 5%, 10%, 15%,
20%, 25%, i.e., the amount of added bacteria suspension is 0.2,1.0,2.0,3.0,4.0,5.0mL, bacteria suspension and inorganic salts culture solution
Total content is 10mL, handles Norfloxacin contaminated soil 10g, is placed in 28 DEG C of cultures, remaining way is identical as application examples 2.Respectively
In the 0th, 5,10,15,20,25,30d sampling extracting, with the residual quantity of high effective liquid chromatography for measuring Ciprofloxacin and calculate it
Degradation rate.
As seen from Figure 6, if other conditions are the same, cultivate 30d after, connect bacterium amount be 1%, 5%, 10%, 15%,
The degradation rate of 20% pair of Ciprofloxacin soil is respectively 38.66%, 48.85%, 41.6%, 36.7%, 37.5%.With inoculation
The raising of amount since growing space and the limited situation hypothallus growth of nutriment can faster reach saturation, and connects bacterium amount too
It is few, although theoretically final increment can be bigger more than bacterium amount than connecing, more incubation times are needed, harmful substance also can be more
It is more, inhibit growth instead.In summary two o'clock, choose 5% for optimal immobilized microorganism connect bacterium amount can arrive preferably drop
Solve effect.
The present invention is inoculated with quinolone antibiotics after the processing of inorganic salts culture solution using agricultural residue (bagasse) as carrier
Efficient degrading bacteria cultivates degradation bacteria, constructs agricultural residue immobilized microorganism system；Prolific immobilization is micro-
Biology adds in the water body or soil polluted by Ciprofloxacin, is uniformly mixed, cultivates at suitable temperature and pH, fixed metaplasia
Object can utilize Ciprofloxacin, so that Ciprofloxacin is degraded, to achieve the purpose that quickly to repair antibiotic polluted-water or soil.
Claims (10)
1. bagasse immobilization microorganism Aspergillus aculeatus, it is characterised in that use bagasse for the fixed degradation bacteria microorganism Aspergillus aculeatus of fixation support
Aspergillus-438。
2. bagasse immobilization microorganism Aspergillus aculeatus according to claim 1, it is characterised in that the bagasse is pre-dried, inoculation
Preceding to pre-process using inorganic salts culture solution, bacteria suspension adds the mixed liquor of inorganic salts culture solution and the amount ratio of bagasse to be 10mL:
1g。
3. bagasse immobilization microorganism Aspergillus aculeatus according to claim 1, it is characterised in that the microorganism Aspergillus aculeatus bacterial strain
Aspergillus-438 first passes through culture in advance before being immobilized onto bagasse carrier and is prepared into bacteria suspension, the bacteria suspension and dry
The amount ratio of dry bagasse is 0.2~5mL ︰ 1g.
4. bagasse immobilization microorganism Aspergillus aculeatus according to claim 3, it is characterised in that by the microorganism Aspergillus aculeatus
Aspergillus-438 bacteria suspension is prepared by following steps culture:
By microbionation after purification on the solid screening and culturing medium in test tube, 28 DEG C are activated 2 days, and the bacterial strain in test tube is used
Normal saline flushing is simultaneously inoculated in the conical flask that volume is 50mL liquid screening medium, in 28 DEG C, the shaking table of 150r/min
It is spare to be placed in 4 DEG C of refrigerators to logarithmic growth phase for upper vortex oscillation culture.
5. bagasse immobilization microorganism Aspergillus aculeatus according to claim 2, it is characterised in that immobilization spine spore is bent on bagasse carrier
The step of mould Aspergillus-438 are as follows:
A, bagasse cleaned into drying, smashed, conical flask is added, high pressure steam sterilization handles 30min, cooling；
B, the inorganic salts culture solution of aseptic process, ultraviolet sterilization 40min are added to conical flask；
C, inoculated and cultured mixes sealing, in 28 DEG C, turns to the microorganism Aspergillus aculeatus Aspergillus-438 bacteria suspension of logarithmic growth phase
Speed is for shaken cultivation in the shaking table of 150r/min to microbes biomass maximum up to bagasse immobilization microorganism Aspergillus aculeatus
Aspergillus-438。
6. a kind of bagasse immobilization microorganism Aspergillus aculeatus of any of claims 1-5 is in quinolone antibiotics pollution amelioration
In application.
7. application of the bagasse immobilization microorganism Aspergillus aculeatus according to claim 6 in quinolone antibiotics pollution amelioration,
It is characterized in that bagasse immobilization microorganism Aspergillus aculeatus Aspergillus-438 quinolone antibiotics Ciprofloxacin in water
In pollution amelioration, the dosage of bagasse immobilization bacterium are as follows: dry bagasse carrier 1g is inoculated with 3.5mL microorganism Aspergillus aculeatus
Aspergillus-438 bacteria suspension administers the Ciprofloxacin contaminant water of 40 times of quality of bagasse, is mixed when processing according to the ratio,
Concussion is handled at 32-43 DEG C, 150r/min.
8. application of the bagasse immobilization microorganism Aspergillus aculeatus according to claim 6 in quinolone antibiotics pollution amelioration,
It is characterized in that bagasse immobilization microorganism Aspergillus aculeatus Aspergillus-438 quinolone antibiotics Ciprofloxacin in the soil
Pollution amelioration in, the dosage of bagasse immobilization bacterium are as follows: dry bagasse carrier 1g is inoculated with 1mL microorganism Aspergillus aculeatus
Aspergillus-438 bacteria suspension, can handle the Ciprofloxacin contaminated soil of 10 times of weight of dry bagasse, according to the ratio when processing
Example is uniformly mixed, and is cultivated at 28 DEG C~43 DEG C, and soil is kept to have certain humidity, water content 40%-60%, to guarantee bacterium
Growth.
9. application of the bagasse immobilization microorganism Aspergillus aculeatus according to claim 6 in quinolone antibiotics pollution amelioration,
It is characterized in that bagasse immobilization microorganism Aspergillus aculeatus Aspergillus-438 quinolone antibiotics Norfloxacin in water
In pollution amelioration, the dosage of bagasse immobilization bacterium are as follows: dry bagasse carrier 1g is inoculated with 3.5mL microorganism Aspergillus aculeatus
Aspergillus-438 bacteria suspension administers the Norfloxacin contaminant water of 40 times of quality of bagasse, is mixed when processing according to the ratio,
Concussion is handled at 37 DEG C, 150r/min.
10. application of the bagasse immobilization microorganism Aspergillus aculeatus according to claim 6 in quinolone antibiotics pollution amelioration,
It is characterized in that bagasse immobilization microorganism Aspergillus aculeatus Aspergillus-438 quinolone antibiotics Norfloxacin in the soil
Pollution amelioration in, the dosage of bagasse immobilization bacterium are as follows: dry bagasse carrier 1g is inoculated with 1mL microorganism Aspergillus aculeatus
Aspergillus-438 bacteria suspension, can handle the Norfloxacin contaminated soil of 10 times of weight of dry bagasse, according to the ratio when processing
Example is uniformly mixed, and is cultivated at 28 DEG C~43 DEG C, and soil is kept to have certain humidity, water content 40%-60%, to guarantee bacterium
Growth.
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Application publication date: 20181211 |