CN106282154B - A kind of preparation method and applications with the co-immobilization mycelium pellet for removing multiple pollutant function - Google Patents
A kind of preparation method and applications with the co-immobilization mycelium pellet for removing multiple pollutant function Download PDFInfo
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- CN106282154B CN106282154B CN201610825353.4A CN201610825353A CN106282154B CN 106282154 B CN106282154 B CN 106282154B CN 201610825353 A CN201610825353 A CN 201610825353A CN 106282154 B CN106282154 B CN 106282154B
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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
- C12N11/16—Enzymes or microbial cells immobilised on or in a biological cell
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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
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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
- C02F2101/308—Dyes; Colorants; Fluorescent agents
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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
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/16—Nature of the water, waste water, sewage or sludge to be treated from metallurgical processes, i.e. from the production, refining or treatment of metals, e.g. galvanic wastes
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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
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/26—Nature of the water, waste water, sewage or sludge to be treated from the processing of plants or parts thereof
- C02F2103/28—Nature of the water, waste water, sewage or sludge to be treated from the processing of plants or parts thereof from the paper or cellulose industry
Abstract
A kind of preparation method and applications with the co-immobilization mycelium pellet for removing multiple pollutant function, are related to a kind of preparation method and applications of co-immobilization mycelium pellet.The present invention is to solve the degradation function of existing mycelium pellet is more single, the bad problem of degradation effect.Method: one, Aspergillus fumigatus spores suspension and bacillus cereus bacteria suspension are prepared；Two, Aspergillus fumigatus spores suspension and bacillus cereus bacteria suspension are taken, is linked into mycelium pellet balling-up culture medium together, shaking table culture to get arrive compound bacteria mycelium pellet.Co-immobilization mycelium pellet is used for lignin degrading, cellulose, hemicellulose, dyestuff and heavy metal ion.The compound bacteria mycelium pellet can not only handle paper waste, it may also be used for processing waste water from dyestuff and effluent containing heavy metal ions, and in alkalinity and the higher system of temperature, still there is preferable treatment effect.The present invention is used for field of waste water treatment.
Description
Technical field
The present invention relates to a kind of preparation method and applications of co-immobilization mycelium pellet.
Background technique
Pulp and paper industry will isolate about 1,400,000,000 tons of celluloses from plant every year, while generating and containing 50,000,000 tons
The sewage of left and right lignin.In lignocellulose, lignin and hemicellulose are combined together in the form of covalent bond, form day
Right barrier contact cellulase can not with cellulose, be to destroy its barrier action using the key of cellulose, and half fiber
Dimension element is easy to remove, so lignin is degraded into for key.The total industrial wastewater discharge of discharge amount Zhan of China's paper waste
The 19% of amount, and discharge of wastewater compliance rate only has 92%, paper-making industry has become one of the emphasis environomental pollution source of China's industry.Print
Dye waste water belongs to industrial wastewater difficult to degrade, have complicated component, water quality and quantity variation greatly, pollutant concentration is high, coloration deeply, can
The features such as biochemical is poor, if will seriously pollute environment without processing direct emission.The use of various dyestuffs, to Dye Removal rate
High request all bring the processing cost of very big difficulty and great number to dyeing waste water factory.Heavy metal pollution to ecological environment and
The influence of human health is on the rise, and the improvement of heavy metal-containing waste water is more and more paid attention to.Copper and lead industrially have
There is very important effect, using extremely wide, the pollution of water body cupric, lead compound has become a kind of serious heavy metal
Pollution.There is strong " three cause " effect to human body and other biologies, to cupric, the processing of lead waste water has become an emphasis class
Topic.
Mainly there are physical method, chemical method and bioanalysis to the processing method of various waste water at present.At physics and chemistry
The energy consumption of reason method is high, and the waste residue or byproduct generated during processing will cause secondary pollution to environment.Both at home and abroad from 20 generation
It records and starts to immobilize the research of microbial technique processing industrial wastewater the end of the eighties, immobilized microorganism technique is passing through
It learns or the means of physics, by free cell or electrodes method in the area of space of restriction, making it keep activity and can recycle.
It with microbe density height, is swift in response, the advantages that microorganism not easily runs off, and product is easily separated, is a kind of low power consuming high efficiency
New technology.This technology is applied to wastewater treatment, is conducive to improve density of the biology in reactor, conducive to consolidating after reaction
Liquid separation, time needed for shortening processing.Mycelium pellet is the sphere being mutually entwined by mycelia, generally fungi, its life
It is strong to deposit ability, sinking speed is fast, is easy to be separated by solid-liquid separation, and it is reusable, have the characteristics that porous surface product is big.And form bacterium
The microorganism of pompon can also normal growth, have the function of biological adsorption and biodegrade etc..
Currently, both at home and abroad for the research of mycelium pellet also in its fermentation industry of exploration and to the processing rank of single waste water
Section.The degradation function of mycelium pellet is more single, and degradation effect is bad.
Summary of the invention
The present invention is to solve the degradation function of existing mycelium pellet is more single, the bad problem of degradation effect provides one
Kind has the preparation method and applications for the co-immobilization mycelium pellet for removing multiple pollutant function.
The present invention have remove multiple pollutant function co-immobilization mycelium pellet preparation method, according to the following steps into
Row:
One, the preparation of bacteria suspension:
Aspergillus fumigatus spores suspension: by the aspergillus fumigatus solid slope being preserved under 4 DEG C of environment take out, with oese by its
It scrapes, is linked into sterile water, it is 1 × 10 that concentration, which is made,6A/mL~9 × 106The Aspergillus fumigatus spores suspension of a/mL；
Bacillus cereus bacteria suspension: the bacillus cereus solid slope being preserved under 4 DEG C of environment is taken out, with inoculation
Ring is scraped, and is linked into sterile water, and it is 1 × 10 that concentration, which is made,6A/mL~5 × 106The bacillus cereus bacterium of a/mL is outstanding
Liquid；
Two, the preparation method of compound bacteria mycelium pellet:
The bacillus cereus bacteria suspension for Aspergillus fumigatus spores suspension and the step 1 preparation for taking step 1 to prepare, connects together
Enter into 100mL mycelium pellet balling-up culture medium, is 120~200r/min in revolving speed, is shaken in the shaking table that temperature is 24~37 DEG C
Cultivate 2~5d.Wherein the volume ratio of Aspergillus fumigatus spores suspension and bacillus cereus bacteria suspension is 1:(1~8).
Aspergillus fumigatus described in step 1 is aspergillus fumigatus YSITB I.Aspergillus fumigatus YSITB I is in 2015 in " aspergillus fumigatus
Lignin research in I bacterial strain screening of YSITB and its degradation wood fibre " (Yuan Junchao, Lv Bao bend the Agriculture In Hubei Province such as honest girl section
Learn .2015,54 (17)) it discloses in article.
Bacillus cereus described in step 1 is bacillus cereus X-10-1-2.Bacillus cereus X-10-1-2 is
In 2006 in " research of two plants of bacillus cellulase-producings " (Yan Hong, Yang Qian, Wang Xiguo chemistry of forest product and industry
.2006,26 (2)) it discloses in article.
Mycelium pellet balling-up culture medium described in step 2 is by 18g/L sucrose, 2.5g/L ammonium tartrate, 2g/L KH2PO4、2g/
L MgSO4·7H2O and distilled water are made, and pH value is adjusted to 5,120 DEG C of sterilizing 20min.
Above-mentioned co-immobilization mycelium pellet is used for lignin degrading, cellulose, hemicellulose and dyestuff and heavy metal ion, institute
Stating dyestuff is Congo red, peacock blue or crystal violet.
Beneficial effects of the present invention:
This method will the aspergillus fumigatus of lignin degrading be trained mycelium pellet as biomass carrier, it is fixed that there is degradation
The bacillus cereus of cellulose ability forms and has both lignin and cellulose degradation ability and the adsorption capacity of mycelium pellet
Compound bacteria mycelium pellet, the two co-incubation, interacts, and collaboration plays a role.It has been investigated that by aspergillus fumigatus and waxy bud
Spore bacillus fixes the compound bacteria mycelium pellet formed, drop of the mycelium pellet that more single aspergillus fumigatus is formed for lignocellulosic altogether
Solution, the absorption of heavy metal ion and the absorption of dyestuff are higher by 10%, 15% and 20% or more respectively, in addition compound bacteria mycelium pellet with
Bacillus cereus is compared, and the degradation rate of cellulose, hemicellulose and lignin also significantly improves.And due to compound bacteria mycelia
Ball secures the bacillus cereus of biodegradable fiber element, and imparts compound bacteria mycelium pellet biodegradable fiber element and hemicellulose
Ability.The study found that compound bacteria pompon respectively can to the highest percent of decolourization of Congo red, peacock blue and crystal violet these three dyestuffs
Up to 97.92%, 95.61% and 88.63%；To Cu2+And Pb2+Highest adsorption rate be respectively 76.96% and 61.32%；To wood
The degradation rate of quality, cellulose and hemicellulose is respectively 63.61%, 47.65% and 63.36%.And pH value be 9 system
Under, the degradation rate of lignin remains to reach 31.47%, and the degradation rate of cellulose and hemicellulose is more than 40%；To Congo red, hole
The percent of decolourization of sparrow indigo plant and crystal violet can respectively reach 79.73%, 70.22% and 76.35%；To Cu2+And Pb2+Adsorption rate
For 50% and 38.67%.And when temperature reaches 40 DEG C, mycelium pellet to the degradation rate of lignin, cellulose and hemicellulose still
37.86%, 32.09% and 37.91% can be respectively reached.This shows that the compound hypha mycelium pellet can not only handle paper waste,
It can also be used to handle waste water from dyestuff and effluent containing heavy metal ions, and in alkalinity and the higher system of temperature, still have preferable
Treatment effect.
Detailed description of the invention
Fig. 1 is compound bacteria mycelium pellet degradation paper waste under different temperatures；
Fig. 2 is compound bacteria mycelium pellet degradation paper waste under different pH；
Fig. 3 is influence of the incubation time to compound bacteria mycelium pellet degradation paper waste；
Fig. 4 is that compound bacteria mycelium pellet adsorbs heavy metal ion under different temperatures；
Fig. 5 is that compound bacteria mycelium pellet adsorbs heavy metal ion under different pH；
Fig. 6 is the influence that incubation time adsorbs heavy metal ion to compound bacteria mycelium pellet；
Fig. 7 is that compound bacteria mycelium pellet adsorbs dyestuff under different temperatures；
Fig. 8 is that compound bacteria mycelium pellet adsorbs Crystal Violet Dye under different pH；
Fig. 9 is the influence that incubation time adsorbs dyestuff to compound bacteria mycelium pellet；
Figure 10 is compound bacteria mycelium pellet photo；
Figure 11 is compound bacteria mycelium pellet inner scanning electromicroscopic photograph；
Figure 12 is the curve that strain density changes over time in compound fermented liquid.
Specific embodiment
The technical solution of the present invention is not limited to the following list, further includes between each specific embodiment
Any combination.
Specific embodiment 1: present embodiment has the preparation for the co-immobilization mycelium pellet for removing multiple pollutant function
Method sequentially includes the following steps:
One, the preparation of bacteria suspension:
Aspergillus fumigatus spores suspension: by the aspergillus fumigatus solid slope being preserved under 4 DEG C of environment take out, with oese by its
It scrapes, is linked into sterile water, it is 1 × 10 that concentration, which is made,6A/mL~9 × 106The Aspergillus fumigatus spores suspension of a/mL；
Bacillus cereus bacteria suspension: the bacillus cereus solid slope being preserved under 4 DEG C of environment is taken out, with inoculation
Ring is scraped, and is linked into sterile water, and it is 1 × 10 that concentration, which is made,6A/mL~5 × 106The bacillus cereus bacterium of a/mL is outstanding
Liquid；
Two, the preparation method of compound bacteria mycelium pellet:
The bacillus cereus bacteria suspension for Aspergillus fumigatus spores suspension and the step 1 preparation for taking step 1 to prepare, connects together
Enter into 100mL mycelium pellet balling-up culture medium, is 120~200r/min in revolving speed, is shaken in the shaking table that temperature is 24~37 DEG C
2~5d is cultivated to get compound bacteria mycelium pellet is arrived.
Wherein aspergillus fumigatus described in step 1 is aspergillus fumigatus YSITB I, and aspergillus fumigatus YSITB I is in 2015 in " cigarette song
Lignin research in mould I bacterial strain screening of YSITB and its degradation wood fibre " (Yuan Junchao, Lv Bao bend the Agriculture In Hubei Province such as honest girl section
Learn .2015,54 (17)) it discloses in article.
Bacillus cereus described in step 1 is bacillus cereus X-10-1-2.The bacillus cereus X-10-
1-2 is in 2006 in " research of two plants of bacillus cellulase-producings " (Yan Hong, Yang Qian, Wang Xiguo chemistry of forest product and work
Industry .2006,26 (2)) it discloses in article.
Specific embodiment 2: the present embodiment is different from the first embodiment in that: aspergillus fumigatus spore in step 1
The concentration of sub- suspension is 3 × 106A/mL~7 × 106A/mL.It is other same as the specific embodiment one.
Specific embodiment 3: the present embodiment is different from the first embodiment in that: aspergillus fumigatus spore in step 1
The concentration of sub- suspension is 5 × 106A/mL.It is other same as the specific embodiment one.
Specific embodiment 4: unlike one of present embodiment and specific embodiment one to three: wax in step 1
The concentration of sample bacillus bacteria suspension is 2 × 106A/mL~4 × 106A/mL.One of other and specific embodiment one to three
It is identical.
Specific embodiment 5: unlike one of present embodiment and specific embodiment one to three: wax in step 1
The concentration of sample bacillus bacteria suspension is 3 × 106A/mL.It is other identical as one of specific embodiment one to three.
Specific embodiment 6: unlike one of present embodiment and specific embodiment one to five: cigarette in step 2
The volume ratio of Aspergillus spore suspension and bacillus cereus bacteria suspension is 1:(1~8).Other and specific embodiment one to five
One of it is identical.
Specific embodiment 7: unlike one of present embodiment and specific embodiment one to five: cigarette in step 2
The volume ratio of Aspergillus spore suspension and bacillus cereus bacteria suspension is 1:(3~5).Other and specific embodiment one to five
One of it is identical.
Specific embodiment 8: unlike one of present embodiment and specific embodiment one to seven: step 2 transfer
Speed is 160r/min.It is other identical as one of specific embodiment one to seven.
Specific embodiment 9: unlike one of present embodiment and specific embodiment one to eight: in step 2
Shake culture 3d in the shaking table that temperature is 28 DEG C.It is other identical as one of specific embodiment one to eight.
Specific embodiment 10: unlike one of present embodiment and specific embodiment one to nine: described in step 2
Mycelium pellet balling-up culture medium is by 18g/L sucrose, 2.5g/L ammonium tartrate, 2g/L KH2PO4、2g/L MgSO4·7H2O and steaming
Distilled water is made, and pH value is adjusted to 5,120 DEG C of sterilizing 20min.It is other identical as one of specific embodiment one to nine.
Specific embodiment 11: present embodiment co-immobilization mycelium pellet is used for lignin degrading, cellulose, hemicellulose
Element, dyestuff and heavy metal ion, the dyestuff are Congo red, peacock blue or crystal violet.
Elaborate below to the embodiment of the present invention, following embodiment under the premise of the technical scheme of the present invention into
Row is implemented, and gives detailed embodiment and specific operating process, but protection scope of the present invention is not limited to following realities
Apply example.
Embodiment:
The present embodiment used medium is as follows:
Mycelium pellet balling-up culture medium: sucrose 18g/L, ammonium tartrate 2.5g/L, KH2PO4 2g/L,MgSO4·7H2O 2g/
L, adds distilled water to be made into 1000mL, and pH value is adjusted to 5,120 DEG C of sterilizing 20min.
Strain store medium: peeled potatoes 200g, glucose 20g/L, KH2PO43g/L, MgSO4·7H2O
1.5g/L, agar 20g/L, trace V B1, add distilled water to be made into 1000mL, pH value is naturally, 120 DEG C of sterilizing 20min.The culture medium
For saving aspergillus fumigatus and bacillus cereus.
One, the preparation of bacteria suspension:
Aspergillus fumigatus bacteria suspension: the aspergillus fumigatus solid slope being preserved under 4 DEG C of environment is taken out, is scraped with oese
Under, it is linked into sterile water, it is 1 × 10 that concentration, which is made,6The spore suspension of a/mL (counting method of blood cell measurement), 4 DEG C of refrigerators save
It is spare.
Bacillus cereus bacteria suspension: the bacillus cereus solid slope being preserved under 4 DEG C of environment is taken out, with inoculation
Ring is scraped, and is linked into sterile water, and it is 5 × 10 that concentration, which is made,6The spore suspension of a/mL (counting method of blood cell measurement), 4 DEG C
Refrigerator saves backup.
Wherein aspergillus fumigatus described in step 1 is aspergillus fumigatus YSITB I, and aspergillus fumigatus YSITB I is in 2015 in " cigarette song
Lignin research in mould I bacterial strain screening of YSITB and its degradation wood fibre " (Yuan Junchao, Lv Bao bend the Agriculture In Hubei Province such as honest girl section
Learn .2015,54 (17)) it discloses in article.
Bacillus cereus described in step 1 is bacillus cereus X-10-1-2.Bacillus cereus X-10-1-2 is
In 2006 in " research of two plants of bacillus cellulase-producings " (Yan Hong, Yang Qian, Wang Xiguo chemistry of forest product and industry
.2006,26 (2)) it discloses in article.
Two, the preparation method of mycelium pellet:
Fungi pellet: taking 5mL aspergillus fumigatus bacteria suspension, is linked into the 250mL cone of the balling-up culture solution of mycelium pellet containing 100mL
It is 160r/min in revolving speed, temperature is shake culture 3d in 30 DEG C of shaking table in shape bottle.
Compound bacteria mycelium pellet: 5mL aspergillus fumigatus bacteria suspension and 20mL bacillus cereus bacteria suspension are taken, is linked into contains together
In the 250mL conical flask of 100mL mycelium pellet balling-up culture solution, it is 160r/min in revolving speed, is shaken in the shaking table that temperature is 28 DEG C
Cultivate 3d.The compound bacteria mycelium pellet photo of preparation is as shown in Figure 10.Compound bacteria mycelium pellet inner scanning electron microscope is as shown in figure 11.
Aspergillus fumigatus and bacillus cereus are added in mycelium pellet balling-up culture solution ferment after, strain density is at any time in compound fermented liquid
The curve of variation is as shown in figure 12.As seen from Figure 12, former with the extension of time, the bacterium amount in fermentation liquid constantly declines
Because being that the equal appendix of thallus is suffered to mycelium pellet, so bacterium amount constantly declines.
Three, degradation of the mycelium pellet to paper waste
Paper waste culture medium is by 100g straw (oven-dry weight), 20gNaOH, 0.5g anthraquinone, 0.795g vulcanized sodium and 580mL
Distilled water composition, is warming up to 125 DEG C, keeps the temperature 2h.
By paper waste culture medium with the dilution proportion of 1:4, pipettes 100mL and be added in the conical flask of 250mL, after sterilizing
The wet mycelium pellet of 5g is added, is placed in isothermal vibration incubator, 28 DEG C, is cultivated under conditions of 160r/min, to be not added at mycelium pellet
The culture medium of reason is blank control, is handled under the same conditions, timing sampling, and the content of lignin in solution is measured.It is described wet
Mycelium pellet is the fungi pellet or compound bacteria mycelium pellet in step 2.
The insoluble lignin of acid: solution is filtered, and adjusts pH to 3-4 with 3% sulfuric acid solution, sediment is dried to perseverance
Weight, the content of the insoluble lignin of acid as in solution.
The molten lignin of acid: pH to 3-4 is adjusted with 3% sulfuric acid solution, stands overnight, is measured in supernatant at 205nm
The content of lignin.
Wherein the quality of lignin is the sum of the insoluble lignin of acid and the molten lignin of acid in solution.
Four, the measurement of cellulose and hemicellulose
0.1g lignin sample is taken, 5mL acetic acid-nitric acid mixed liquor is added, heats 20min, cooled and filtered in boiling water bath.
1mL filtrate is taken, 4mL orcinol reagent is added, 100 DEG C of heat preservation 15min survey OD value at 660nm.It is found out by xylose standard curve
Sugar amount is multiplied by the content that coefficient 0.9 is hemicellulose.Twice with acetone washing, 60 DEG C of dryings are placed in beaker to constant weight to filter residue
Middle addition 5mL72% sulfuric acid, 20 DEG C of hydrolysis 3h add water 45mL, ambient temperature overnight, and next day filtering takes 2mL filtrate that 5mL anthrone is added
Reagent, 100 DEG C of heat preservation 10min survey OD value at 620nm, find out sugar amount by glucose standard curve, being multiplied by coefficient 0.9 is
The content of cellulose.
Before inoculation that degradation rate calculates: degradation rate (DR)=(be inoculated with before certain component content-inoculation after certain component content)/certain
Component content × 100%
Acetic acid described in step 4-nitric acid mixed liquor is made of mixed 10mL concentrated nitric acid and 100mL80% acetic acid (v/v)
Close liquid.
Five, measurement of the mycelium pellet to Dye Adsorption ability
Dyestuff culture solution includes congo red culture medium, malachite green dyestuff culture medium and Crystal Violet Dye culture medium,
Congo red culture medium: Congo red 100mg adds distilled water to be made into 1000mL, and pH value is naturally, 120 DEG C of sterilizings
20min。
Malachite green dyestuff culture medium: malachite green 100mg adds distilled water to be made into 1000mL, and pH value is naturally, 120 DEG C go out
Bacterium 20min.
Crystal Violet Dye culture medium: crystal violet 100mg adds distilled water to be made into 1000mL, and pH value is naturally, 120 DEG C of sterilizings
20min。
The wet mycelium pellet of 5g is taken, is added in the 250mL conical flask of the dyestuff culture solution containing 100mL, the concentration of dyestuff is
100mg/L, the type of dyestuff are Congo red, malachite green, crystal violet.It is placed in isothermal vibration incubator, 28 DEG C, 160r/min
Under conditions of cultivate.Timing sampling, the supernatant after taking centrifugal filtration, in maximum absorption wave strong point UV, visible light spectrophotometric
Measure its absorbance A1, not to be inoculated with the absorbance A of the dyestuff culture medium of mycelium pellet0For control, percent of decolourization is calculated.The wherein the Congo
Red, malachite green, crystal violet maximum absorption wavelength be respectively 506nm, 617.4nm and 586nm.The wet mycelium pellet is step
Fungi pellet or compound bacteria mycelium pellet in two.
The calculation formula of percent of decolourization is as follows:
Percent of decolourization (%)=[(A0-A1)/A1] × 100%
Experimental result is as follows:
1, the performance of single fungi pellet and compound bacteria mycelium pellet processing various wastewater compares
Paper waste culture medium is by 100g straw (oven-dry weight), 20gNaOH, 0.5g anthraquinone, 0.795g vulcanized sodium and 580mL
Distilled water composition, is warming up to 125 DEG C, keeps the temperature 2h
5g fungi pellet and 5g compound bacteria mycelium pellet are inoculated in paper waste culture medium, heavy metal ion culture respectively
In base and dyestuff culture medium, the liquid amount of three kinds of culture mediums is 100mL (250mL conical flask), is 28 DEG C in temperature, revolving speed is
Shake culture 3d in the shaking table of 160r/min, sampling, measure two kinds of mycelium pellets to the degradation rate of paper waste, to heavy metal ion
Adsorption rate and percent of decolourization to dyestuff.Two kinds of mycelium pellets are shown in Table 1 to the processing result of waste water.
The performance that 1 fungi pellet of table and compound bacteria mycelium pellet handle various waste water compares
| Compound bacteria mycelium pellet | Fungi pellet | |
| Cellulose degradation rate/% | 39.05 | 2.38 |
| Hemicellulose degradation rate/% | 50.9 | 4.16 |
| Lignin degradation rate/% | 51.61 | 34.05 |
| Cu adsorption rate/% | 65.96 | 38.27 |
| Pb adsorption rate/% | 52.33 | 34.06 |
| Congo red percent of decolourization/% | 92.74 | 69.51 |
| Peacock blue percent of decolourization/% | 91.61 | 71.13 |
| Crystal violet percent of decolourization/% | 84.23 | 62.33 |
As can be seen from Table 1, for the degradation of paper waste, fungi pellet is to lignin, cellulose, hemicellulose
Degradation rate is respectively 34.05%, 2.38%, 4.16%.Compound bacteria mycelium pellet is respectively 51.61% to its degradation rate, 50.9%,
39.05%.It can thus be seen that fungi pellet is hardly degraded ability, compound bacteria mycelia to cellulose and hemicellulose
Degradation of the ball compared with fungi pellet for lignin, degradation rate are higher by 10% or more.Absorption for heavy metal ion, fungi bacterium
Pompon is to Cu2+And Pb2+Adsorption rate be respectively 38.27% and 34.06%.Compound bacteria mycelium pellet is to Cu2+And Pb2+Adsorption rate
Respectively 65.96% and 52.33%.Absorption of the more single fungi pellet of compound bacteria mycelium pellet to heavy metal ion, adsorption rate
Improve 15% or so.Processing for waste water from dyestuff, fungi pellet is to Congo red, peacock blue, crystal violet percent of decolourization point
It Wei 69.51%, 71.13%, 62.33%.Compound bacteria mycelium pellet is respectively 92.74% to the percent of decolourization of three of the above dyestuff,
91.16%, 84.23%.Percent of decolourization is higher than 20% or more fungi pellet.
In addition, bacillus cereus X10-1-2 and compound bacteria mycelium pellet are inoculated into wheat bran culture by identical inoculum concentration
In base and straw culture medium, in 30 DEG C, revolving speed is shake culture 5d in the shaking table of 160r/min, measures bacillus cereus X10-
1-2 and compound bacteria mycelium pellet the results are shown in Table 2 to the degradation rate of cellulose, hemicellulose, lignin in wheat bran and straw.
Wheat bran medium is by 3.0g Na2HPO4、2.0g(NH4)2SO4, 0.5g urea, 0.5g MgSO4·7H2O、0.5g
CaCl2、7.5mg FeSO4·7H2O、2.5mg MnSO4·H2O and 2.0mg ZnSO4It is made into 1000mL, adds 2% wheat bran,
0.1% peptone, 1% yeast powder, adjusting pH value is 7.0~7.2.
Straw culture medium is by 3.0g Na2HPO4、2.0g(NH4)2SO4, 0.5g urea, 0.5g MgSO4·7H2O、0.5g
CaCl2、7.5mg FeSO4·7H2O、2.5mg MnSO4·H2O and 2.0mg ZnSO4It is made into 1000mL, adds 2% straw,
0.1% peptone, 1% yeast powder, adjusting pH value is 7.0~7.2.
The performance that 2 bacillus cereus X10-1-2 of table and compound bacteria mycelium pellet handle various waste water compares
From the above data, the performance of compound bacteria mycelium pellet is superior to single fungi pellet, while also superior to single
Bacillus cereus X10-1-2.Compound bacteria mycelium pellet is a kind of two microorganisms immobilization system, simultaneously containing lignin degrading
The bacterium of fungi and degraded cellulose, two strains have complementary performance, play the role of mutually promoting, and both have lignin
Degradation capability has cellulose degradation vigor again.
2, compound bacteria mycelium pellet handles the performance study of various waste water
1. handling paper waste
The performance of compound bacteria mycelium pellet degradation paper waste under different temperatures:
5g compound bacteria mycelium pellet is inoculated in the 250mL conical flask of the culture medium of paper waste containing 100mL, is in revolving speed
160r/min, temperature are respectively shake culture in 24 DEG C, 28 DEG C, 30 DEG C, 34 DEG C, 37 DEG C and 40 DEG C of shaking table, are sampled every 2d,
Measure the degradation rate of lignocellulosic.It measures the maximum value that ligocellulose degradation at each temperature leads and sees Fig. 1, in figure ◆ indicate wood
The degradation rate of quality, ■ indicate the degradation rate of cellulose, the degradation rate of ▲ expression hemicellulose.
As shown in Figure 1, the preference temperature of mycelium pellet lignin degrading is 28 DEG C~34 DEG C, the lignin when temperature is 28 DEG C
Degradation rate highest, up to 58.01%.In 30 DEG C and 34 DEG C, the degradation rate of cellulose and hemicellulose reaches highest, respectively
For 45.39% and 57.45%.When temperature is 40 DEG C, the degradation rate of hemicellulose remains to reach 37.91%, illustrates in high temperature
In the environment of, mycelium pellet still has good degradation to paper waste.
The performance of compound bacteria mycelium pellet degradation paper waste under different pH:
5g compound bacteria mycelium pellet is inoculated in the 250mL conical flask of the culture medium of paper waste containing 100mL, adjusts culture medium
PH be respectively 2,3,5,7,9,11 and 12, be 160r/min in revolving speed, temperature is shake culture in 28 DEG C of shaking table, every 2d
Sampling, measures the degradation rate of lignocellulosic, measures the maximum value that ligocellulose degradation leads under each pH value and sees Fig. 2, in figure ◆
Indicate that the degradation rate of lignin, ■ indicate the degradation rate of cellulose, the degradation rate of ▲ expression hemicellulose.
As shown in Figure 2, when pH value is less than 5, the degradation rate of lignin is respectively less than 20%, illustrates that the environment of meta-acid can inhibit
The generation of lignin-degrading enzymes is unfavorable for the degradation of lignin in papermaking wastewater.Lignin degradation rate highest when pH is 5, it is reachable
To 61.38%.With the increase of pH, Lignin degradation rate decreases, and under the system that pH is 9, the degradation rate of lignin is remained to
Reach 31.47%, cellulose and hemicellulose degradation rate can maintain 40% or more under 5~11 system, illustrate inclined
Under acid and alkalinity environment, compound bacteria mycelium pellet can still provide for degrading well to papermaking wastewater.
The performance of compound bacteria mycelium pellet degradation paper waste under different time:
5g compound bacteria mycelium pellet is inoculated in the 250mL conical flask of the culture medium of paper waste containing 100mL, is in revolving speed
160r/min, temperature are shake culture in 28 DEG C of shaking table, sample daily, measure the degradation rate of lignocellulosic, as a result see figure
3, in figure ◆ indicate that the degradation rate of lignin, ■ indicate the degradation rate of cellulose, × indicate the degradation rate of hemicellulose.
As seen from Figure 3, with the extension of incubation time, mycelium pellet first increases the degradation rate presentation of lignin to drop afterwards
Low trend is big in system when beginning this is because lignin is mainly to be made of the molten lignin of acid and the insoluble lignin of acid
Molecule lignin segment gradually decreases, and degradation rate gradually rises, and when 144h is arrived in culture, degradation rate reaches up to 63.61%.
However, the macromolecular gluco in the molten lignin of acid can be degraded to small molecule segment by mycelium pellet, therefore acid is not in degradation process
The content of molten lignin will increase, and lignin completely removes content reduction, and total Lignin degradation rate is reduced.Due to mycelium pellet
With very strong degradation capability, with the degradation of small molecule lignin segment, Lignin degradation rate gradually rises again.Compound bacteria bacterium
Pompon also has very high degradation capability to cellulose and hemicellulose, and in 72h and 60h, highest degradation rate is respectively
49.05% and 63.36%.
Processing paper waste is recycled in mycelium pellet:
The stability and recycling effect for investigating compound bacteria mycelium pellet, 5g compound bacteria mycelium pellet is inoculated in containing 100mL
It is 160r/min in revolving speed in the 250mL conical flask of paper waste culture medium, temperature is shake culture 5d in 28 DEG C of shaking table,
It completes after recycling for the first time, this compound bacteria mycelium pellet is linked into new wastewater medium, handles 5d at identical conditions.
So circulation 4 times, the results are shown in Table 3.
Processing paper waste result is recycled in 3 compound bacteria mycelium pellet of table
As shown in Table 2, in the circulation of first time, mycelium pellet is fine to the degradation effect of lignocellulosic, lignin, fibre
Tieing up plain, hemicellulose degradation rate is respectively 51.27%, 41.58%, 57.1%, in second of circulation to third time, bacterium
Pompon declines the degradation rate of lignocellulosic, and mycelia ball surface becomes rough, and sphere color becomes dark-brown, but right
The degradation rate of three kinds of lignocellulosics remains to reach 20% or more.When being recycled to the 4th time, mycelium pellet just starts
The phenomenon that existing self-dissolving.This shows that mycelium pellet can maintain activity for a long time, the processing waste water that can continue.
2. processing of the compound bacteria mycelium pellet to effluent containing heavy metal ions
The performance of compound bacteria mycelium pellet absorption heavy metal ion under different temperatures:
Heavy metal ion culture medium includes Cu2+Culture medium and Pb2+Culture medium,
Cu2+Culture medium: plumbi nitras 79.93mg adds distilled water to be made into 1000mL, and pH value is naturally, 120 DEG C of sterilizing 20min.
Pb2+Culture medium: copper sulphate 195.32mg adds distilled water to be made into 1000mL, and pH value is naturally, 120 DEG C of sterilizing 20min.
5g compound bacteria mycelium pellet is inoculated in the 250mL conical flask of the culture medium of heavy metal ion containing 100mL, is in revolving speed
160r/min, temperature is respectively shake culture in 20 DEG C, 24 DEG C, 28 DEG C, 30 DEG C, 34 DEG C, 37 DEG C and 40 DEG C of shaking table, every 1d
Sampling, measures heavy metal ion adsorbed rate.The maximum value for measuring rate heavy metal ion adsorbed at each temperature is shown in Fig. 4, in figure ◆ table
Show that Cu, ■ indicate Pb.
As shown in Figure 4, the temperature investigated in range has a certain impact to the absorption of heavy metal ion.At 28 DEG C~34 DEG C
Adsorption effect in range is all fine, and compound bacteria mycelium pellet is to Cu2+Adsorption rate reach highest at 28 DEG C, to Pb2+Absorption
Rate reaches highest at 30 DEG C, and respectively 63.73% and 57.83%.When temperature is 20 DEG C and 40 DEG C, the suction of compound bacteria mycelium pellet
Attached effect is worst, illustrates too high or too low for temperature can all influence absorption of the compound bacteria mycelium pellet to heavy metal ion.
The performance of compound bacteria mycelium pellet absorption heavy metal ion under different pH:
5g compound bacteria mycelium pellet is inoculated in the 250mL conical flask of the culture medium of heavy metal ion containing 100mL, adjusts culture
The pH of base is respectively 2,3,5,7,9,11 and 12, is 160r/min in revolving speed, and temperature is shake culture in 28 DEG C of shaking table, every
1d sampling, measures heavy metal ion adsorbed rate.The maximum value for measuring heavy metal ion adsorbed rate under each pH value is shown in Fig. 5, in figure ◆
Indicate that Cu, ■ indicate Pb.
The pH value of solution can influence the chemical state of metal ion and mycelium pellet cell surface simultaneously, study pH value to mycelia
The influence of ball absorption heavy metal ion has a very important significance.As shown in Figure 5, when pH value is less than 5, compound bacteria mycelium pellet
To Cu2+And Pb2+Adsorption rate it is all very low, this is because when pH is smaller, proton occupies most of absorption of mycelia ball surface
Site reduces the adsorption efficiency of heavy metal ion.Adsorption effect of pH value in the range of 5~9 is all fine, when pH value is 5
When, Cu2+And Pb2+Adsorption rate reach maximum, respectively 73.21% and 59.29%.OH when pH value is greater than 9, in solution-
Ion can occur Competition with metal ion, then adsorption rate be caused to reduce with the functional groups in mycelia ball surface.Knot
Fruit shows that compound bacteria mycelium pellet has good adsorption effect under meta-acid and alkaline environment to heavy metal ion.
The performance of compound bacteria mycelium pellet absorption heavy metal ion under different time:
5g compound bacteria mycelium pellet is inoculated in the 250mL conical flask of the culture medium of heavy metal ion containing 100mL, is in revolving speed
160r/min, temperature are shake culture in 28 DEG C of shaking table.When starting measurement, is sampled every 30min, measure heavy metal ion
Adsorption rate, is as a result shown in Fig. 6, in figure ◆ indicates that Cu, ■ indicate Pb.
It will be appreciated from fig. 6 that compound bacteria mycelium pellet is to Cu2+And Pb2+Be adsorbed in 10 hours and just respectively reach
63.99% and 37.53%, the concentration of heavy metal ion declines quickly, this is because this absorption phase in solution in a short time
For the surface quick adsorption stage, various active groups and heavy metal ion ligand complex on mycelium pellet cell wall are adsorbed.With
In adsorption time afterwards, the concentration of heavy metal ion in solution slowly declines up to adsorption equilibrium, this is because with adsorbance
Increase, mycelium pellet cell wall reached saturation to the absorption of heavy metal ion, and free heavy metal ion is thin to enter
Intracellular, suffered resistance just will increase, therefore the time for reaching adsorption saturation will be very long.In 48h and 72h, compound bacteria bacterium
Pompon is to Cu2+And Pb2+Adsorption rate reach maximum, respectively 76.96% and 61.32%.
3. the processing to a variety of waste water from dyestuff
The performance of compound bacteria mycelium pellet absorption dyestuff under different temperatures:
Dyestuff culture solution includes congo red culture medium, malachite green dyestuff culture medium and Crystal Violet Dye culture medium,
Congo red culture medium: Congo red 100mg adds distilled water to be made into 1000mL, and pH value is naturally, 120 DEG C of sterilizings
20min。
Malachite green dyestuff culture medium: malachite green 100mg adds distilled water to be made into 1000mL, and pH value is naturally, 120 DEG C go out
Bacterium 20min.
Crystal Violet Dye culture medium: crystal violet 100mg adds distilled water to be made into 1000mL, and pH value is naturally, 120 DEG C of sterilizings
20min。
5g compound bacteria mycelium pellet is inoculated in the 250mL conical flask of the culture medium of dyestuff containing 100mL, in revolving speed be 160r/
Min, temperature are respectively shake culture in 20 DEG C, 24 DEG C, 28 DEG C, 30 DEG C, 34 DEG C and 40 DEG C of shaking table, are sampled every 12h, measurement
The percent of decolourization of dyestuff, the maximum value for measuring rate dye decolored at each temperature are shown in Fig. 7, in figure ◆ indicate Congo red, ■ indicates peacock
Indigo plant, ▲ indicate crystal violet.
As shown in Figure 7, temperature has a great impact to compound bacteria mycelium pellet absorption dyestuff, when temperature is at 20 DEG C~40 DEG C
When, percent of decolourization has the trend of a first increases and then decreases as the temperature increases.It is multiple within the temperature range of 28 DEG C~34 DEG C
Combined bacteria mycelium pellet is all fine to the decolorizing effect of three kinds of dyestuffs.When temperature reaches 28 DEG C, compound bacteria mycelium pellet to peacock blue and
Congo red adsorption effect is best, and percent of decolourization is up to 94.75% and 96.43%；When 30 DEG C of temperature, compound bacteria mycelium pellet is to knot
The adsorption effect of crystalviolet is best, and percent of decolourization can reach 81.63%.When temperature reaches 40 DEG C, compound bacteria mycelium pellet contaminates three kinds
The percent of decolourization of material is all 30% or so.It is possible thereby to infer, the variation of temperature will affect the secretion of lignocellulolyticenzymes and change
Become the structure and property of enzyme, the excessively high compound bacteria mycelium pellet that can reduce of temperature is to the percent of decolourization of dyestuff.
The performance of compound bacteria mycelium pellet absorption dyestuff under different pH:
5g compound bacteria mycelium pellet is inoculated in 100mL (250mL conical flask) dyestuff culture medium, pH points of culture medium are adjusted
Not Wei 2,3,5,7,9,11 and 12, be 160r/min in revolving speed, temperature is shake culture in 28 DEG C of shaking table, sampled every 12h,
The percent of decolourization for measuring dyestuff, the maximum value for measuring dye decolored rate under each PH are shown in Fig. 8, in figure ◆ indicate Congo red, ■ indicates hole
Sparrow is blue, ▲ indicate crystal violet.
Compound bacteria mycelium pellet mainly relies on degradation the decoloration of dyestuff the modes such as catalysis oxidation, the reduction of enzyme system broken
The unsaturated conjugated key and color development system of bad dyestuff, pH has a great impact to enzyme's reaction speeding, only in Optimal pH condition
Under, enzyme can just show best catalytic activity.As shown in Figure 8, for pH in the range of 5~9, compound bacteria mycelium pellet contaminates three kinds
The percent of decolourization of material is all up to 70% or more.When pH be 5 when, compound bacteria mycelium pellet to Congo red and peacock blue decolorizing effect most
Good, percent of decolourization can reach 97.87% and 94.55%；When pH is 7, compound bacteria mycelium pellet is best to the decolorizing effect of crystal violet,
Percent of decolourization is 83.67%.The result shows that compound bacteria mycelium pellet has good absorption in the environment of inclined acid and alkaline to dyestuff
Effect.
The performance of compound bacteria mycelium pellet absorption dyestuff under different time:
5g compound bacteria mycelium pellet is inoculated in the 250mL conical flask of the culture medium of dyestuff containing 100mL, in revolving speed be 160r/
Min, temperature is shake culture in 28 DEG C of shaking table, when starting measurement, samples every 30min, measures the percent of decolourization of dyestuff, as a result
Fig. 9 is seen, in figure ◆ indicate Congo red, ■ indicates peacock blue, ▲ indicate crystal violet.
As shown in Figure 9, compound bacteria mycelium pellet increases the adsorbance of three kinds of dyestuffs very fast within initial a period of time
Speed then gradually slows down until adsorption equilibrium.For these three dyestuffs of Congo red, peacock blue and crystal violet, within initial 12h
Percent of decolourization just can reach 70% or more, decolorization rate is relatively high.In 6h, compound bacteria mycelium pellet to Congo red percent of decolourization most
Height can reach 97.92%.When cultivating 48h, for compound bacteria mycelium pellet to peacock blue, the adsorption effect of crystal violet is best, and highest is de-
Color rate is respectively 95.61% and 88.63%.The result shows that compound bacteria mycelium pellet achieves well the decoloration of three kinds of dyestuffs
Effect.
Claims (9)
1. a kind of preparation method with the co-immobilization mycelium pellet for removing multiple pollutant function, it is characterised in that this method packet
Include following steps:
One, the preparation of bacteria suspension:
Aspergillus fumigatus spores suspension: the aspergillus fumigatus solid slope being preserved under 4 DEG C of environment is taken out, is scraped with oese
Under, it is linked into sterile water, it is 1 × 10 that concentration, which is made,6A/mL~9 × 106The Aspergillus fumigatus spores suspension of a/mL；
Bacillus cereus bacteria suspension: the bacillus cereus solid slope being preserved under 4 DEG C of environment is taken out, will with oese
It is scraped, and is linked into sterile water, and it is 1 × 10 that concentration, which is made,6A/mL~5 × 106The bacillus cereus bacteria suspension of a/mL；
Two, the preparation method of compound bacteria mycelium pellet:
The bacillus cereus bacteria suspension for Aspergillus fumigatus spores suspension and the step 1 preparation for taking step 1 to prepare, is linked into together
It is 120~200r/min in revolving speed, temperature is shake culture 2 in 24~37 DEG C of shaking table in 100mL mycelium pellet balling-up culture medium
~5d to get arrive compound bacteria mycelium pellet；
Wherein aspergillus fumigatus described in step 1 is aspergillus fumigatus YSITB I,
Bacillus cereus described in step 1 is bacillus cereus X-10-1-2；
The volume ratio of Aspergillus fumigatus spores suspension and bacillus cereus bacteria suspension is 1:(1~8 in step 2).
2. a kind of preparation side with the co-immobilization mycelium pellet for removing multiple pollutant function according to claim 1
Method, it is characterised in that the concentration of Aspergillus fumigatus spores suspension is 3 × 10 in step 16A/mL~7 × 106A/mL.
3. a kind of preparation side with the co-immobilization mycelium pellet for removing multiple pollutant function according to claim 1
Method, it is characterised in that the concentration of bacillus cereus bacteria suspension is 2 × 10 in step 16A/mL~4 × 106A/mL.
4. a kind of preparation side with the co-immobilization mycelium pellet for removing multiple pollutant function according to claim 1
Method, it is characterised in that the volume ratio of Aspergillus fumigatus spores suspension and bacillus cereus bacteria suspension is 1:(3~5 in step 2).
5. a kind of preparation side with the co-immobilization mycelium pellet for removing multiple pollutant function according to claim 1
Method, it is characterised in that revolving speed is 160r/min in step 2.
6. a kind of preparation side with the co-immobilization mycelium pellet for removing multiple pollutant function according to claim 1
Method, it is characterised in that in temperature be shake culture 3d in 28 DEG C of shaking table in step 2.
7. a kind of preparation side with the co-immobilization mycelium pellet for removing multiple pollutant function according to claim 1
Method, it is characterised in that mycelium pellet balling-up culture medium described in step 2 is by 18g/L sucrose, 2.5g/L ammonium tartrate, 2g/L
KH2PO4、2g/L MgSO4.7H2O and distilled water are made, and pH value is adjusted to 5,120 DEG C of sterilizing 20min.
8. the application of the co-immobilization mycelium pellet of the method as described in claim 1 preparation, it is characterised in that co-immobilization mycelia
Ball is used for lignin degrading, cellulose, hemicellulose, dyestuff and heavy metal ion.
9. application according to claim 8, it is characterised in that the dyestuff is Congo red, peacock blue or crystal violet.
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