CN106282154A - A kind of preparation method and applications with the co-immobilization mycelium pellet removing multiple pollutant function - Google Patents
A kind of preparation method and applications with the co-immobilization mycelium pellet removing multiple pollutant function Download PDFInfo
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- CN106282154A CN106282154A CN201610825353.4A CN201610825353A CN106282154A CN 106282154 A CN106282154 A CN 106282154A CN 201610825353 A CN201610825353 A CN 201610825353A CN 106282154 A CN106282154 A CN 106282154A
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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 removing multiple pollutant function, relate to the preparation method and applications of a kind of co-immobilization mycelium pellet.The present invention is to solve that the degradation function of existing mycelium pellet is the most single, the problem that degradation effect is the best.Method: one, prepare Aspergillus fumigatus spores suspension and bacillus cereus bacteria suspension；Two, taking Aspergillus fumigatus spores suspension and bacillus cereus bacteria suspension, be linked into together in mycelium pellet balling-up culture medium, shaking table is cultivated, and i.e. obtains compound bacteria mycelium pellet.Co-immobilization mycelium pellet is for lignin degrading, cellulose, hemicellulose, dyestuff and heavy metal ion.This compound bacteria mycelium pellet can not only process paper waste, it may also be used for processes waste water from dyestuff and effluent containing heavy metal ions, and in the system that alkalescence and temperature are higher, still has preferable treatment effect.The present invention is used for field of waste water treatment.
Description
Technical field
The present invention relates to the preparation method and applications of a kind of co-immobilization mycelium pellet.
Background technology
Pulp and paper industry to isolate about 1,400,000,000 tons of celluloses every year from plant, produces containing 50,000,000 tons simultaneously
The sewage of left and right lignin.In lignocellulose, lignin combines with covalent bond form with hemicellulose, forms sky
Right barrier, makes cellulase cannot contact with cellulose, and utilize cellulose it is critical only that its barrier action of destruction, and half is fine
Dimension element easily removes, so being degraded into for key of lignin.The discharge capacity of China's paper waste accounts for total industrial wastewater discharge
The 19% of amount, and discharge of wastewater compliance rate only has 92%, papermaking had become one of emphasis environomental pollution source of China's industry already.Print
Dye waste water belongs to the industrial wastewater of difficult degradation, there is complicated component, water quality and quantity changes greatly, pollutant levels are high, colourity deeply, can
The features such as biochemical difference, if the most treated direct discharge, by serious environment pollution.The use of various dyestuffs, to Dye Removal rate
High request bring the processing cost of the biggest difficulty and great number all to dyeing waste water factory.Heavy metal pollution to ecological environment and
The impact 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
Having very important effect, its application is extremely wide, and water body cupric, the pollution of lead compound have become a kind of serious heavy metal
Pollute.Have strong " three cause " effect to human body and other biology, to cupric, the process of lead waste water has become an emphasis class
Topic.
At present the processing method of various waste water mainly there are physical method, chemical method and bioanalysis.At physics and chemistry
Reason method power consumption height, environment can be caused secondary pollution by the waste residue or the side-product that produce in processing procedure.Both at home and abroad from 20 generation
Recording and proceed by the research of immobilized microorganism technique process industrial wastewater the end of the eighties, immobilized microorganism technique is by changing
Learn or the means of physics, by free cell or electrodes method in the area of space limited so that it is keep activity also can recycle.
Having microbe density high, be swift in response, the advantages such as microorganism not easily runs off, and product is easily separated, are a kind of low power consuming high efficiency
New technique.This technology is applied to waste water and processes, and is conducive to improving biological density in reactor, the most reacted solid
Liquid separates, the time needed for shortening process.Mycelium pellet is the spheroid being mutually entwined by mycelia, generally fungus, its life
Ability of depositing is strong, and sedimentation velocity is fast, it is easy to solid-liquid separation, repeatable utilization, has porous surface and amasss big feature.And form bacterium
The microorganism of pompon can also normal growth, there is the function such as biological adsorption and biodegradation.
At present, both at home and abroad mycelium pellet is studied also in exploring its fermentation industry and the process rank to single waste water
Section.The degradation function of mycelium pellet is the most single, and degradation effect is the best.
Summary of the invention
The present invention is to solve that the degradation function of existing mycelium pellet is the most single, the problem that degradation effect is the best, it is provided that
Plant the preparation method and applications with the co-immobilization mycelium pellet removing multiple pollutant function.
The present invention has the preparation method of the co-immobilization mycelium pellet removing multiple pollutant function, enters according to the following steps
OK:
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, with inoculating loop by it
Scraping, be linked in sterilized water, making concentration is 1 × 106Individual/mL～9 × 106The Aspergillus fumigatus spores suspension of individual/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 in sterilized water, and making concentration is 1 × 106Individual/mL～5 × 106The bacillus cereus bacterium of individual/mL is hanged
Liquid；
Two, the preparation method of compound bacteria mycelium pellet:
Take Aspergillus fumigatus spores suspension and the bacillus cereus bacteria suspension of step one preparation of step one preparation, connect together
Entering in 100mL mycelium pellet balling-up culture medium, be 120～200r/min in rotating speed, temperature is to shake in the shaking table of 24～37 DEG C
Cultivate 2～5d.Wherein Aspergillus fumigatus spores suspension is 1:(1～8 with the volume ratio of bacillus cereus bacteria suspension).
Aspergillus fumigatus described in step one is Aspergillus fumigatus YSITB I.Aspergillus fumigatus YSITB I in 2015 " Aspergillus fumigatus
Lignin research in YSITB I bacterial strain screening and degraded wood fibre thereof " (Yuan Junchao, Lv Bao bend honest girl etc. Agriculture In Hubei Province section
Learn .2015,54 (17)) disclosed in article.
Bacillus cereus described in step one is bacillus cereus X-10-1-2.Bacillus cereus X-10-1-2 is
In 2006 " researchs of two strain bacillus cellulase-producings " (Yan Hong, Yang Qian, Wang Xiguo. chemistry of forest product with industry
.2006,26 (2)) disclosed 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 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 the Aspergillus fumigatus of lignin degrading will can be trained mycelium pellet as biomass carrier, and fixing have degraded
The bacillus cereus of cellulose ability, formation has the absorbability of lignin and cellulose degradation ability and mycelium pellet concurrently
Compound bacteria mycelium pellet, the two co-cultivation, interact, work in coordination with and play a role.It has been investigated that, by Aspergillus fumigatus and waxy bud
The spore bacillus fixing compound bacteria mycelium pellet formed altogether, the mycelium pellet that more single Aspergillus fumigatus is formed is for the fall of lignocellulose
Solve, the absorption of the absorption of heavy metal ion and dyestuff exceeds 10%, 15% and more than 20% respectively, additionally 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, imparts again compound bacteria mycelium pellet biodegradable fiber element and hemicellulose
Ability.Research finds, compound bacteria pompon respectively can to the highest percent of decolourization of Congo red, peacock blue and crystal violet these three dyestuff
Reach 97.92%, 95.61% and 88.63%；To Cu2+And Pb2+High 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 be the system of 9 at pH value
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
Blue and crystal violet the percent of decolourization of passeris montani saturati can respectively reach 79.73%, 70.22% and 76.35%；To Cu2+And Pb2+Adsorption rate
It is 50% and 38.67%.And when temperature reaches 40 DEG C, mycelium pellet is to the degradation rate of lignin, cellulose and hemicellulose still
37.86%, 32.09% and 37.91% can be respectively reached.This shows that this compound hypha mycelium pellet can not only process paper waste,
Can be additionally used in process waste water from dyestuff and effluent containing heavy metal ions, and in the system that alkalescence and temperature are higher, still have preferably
Treatment effect.
Accompanying drawing explanation
Fig. 1 is compound bacteria mycelium pellet degraded paper waste under different temperatures；
Fig. 2 is compound bacteria mycelium pellet degraded paper waste under different pH；
Fig. 3 is the incubation time impact on compound bacteria mycelium pellet degraded paper waste；
Fig. 4 is compound bacteria mycelium pellet Adsorption of Heavy Metal Ions under different temperatures；
Fig. 5 is compound bacteria mycelium pellet Adsorption of Heavy Metal Ions under different pH；
Fig. 6 is the incubation time impact on compound bacteria mycelium pellet Adsorption of Heavy Metal Ions；
Fig. 7 is compound bacteria mycelium pellet absorbing dye under different temperatures；
Fig. 8 is compound bacteria mycelium pellet absorption Crystal Violet Dye under different pH；
Fig. 9 is the incubation time impact on compound bacteria mycelium pellet absorbing dye；
Figure 10 is compound bacteria mycelium pellet photo；
Figure 11 is compound bacteria mycelium pellet inner scanning electromicroscopic photograph；
Figure 12 is the time dependent curve of strain density in compound bacteria-fermented liquid.
Detailed description of the invention
Technical solution of the present invention is not limited to act detailed description of the invention set forth below, also includes between each detailed description of the invention
Combination in any.
Detailed description of the invention one: present embodiment has the preparation of the co-immobilization mycelium pellet removing multiple pollutant function
Method, sequentially includes the 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, with inoculating loop by it
Scraping, be linked in sterilized water, making concentration is 1 × 106Individual/mL～9 × 106The Aspergillus fumigatus spores suspension of individual/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 in sterilized water, and making concentration is 1 × 106Individual/mL～5 × 106The bacillus cereus bacterium of individual/mL is hanged
Liquid；
Two, the preparation method of compound bacteria mycelium pellet:
Take Aspergillus fumigatus spores suspension and the bacillus cereus bacteria suspension of step one preparation of step one preparation, connect together
Entering in 100mL mycelium pellet balling-up culture medium, be 120～200r/min in rotating speed, temperature is to shake in the shaking table of 24～37 DEG C
Cultivate 2～5d, i.e. obtain compound bacteria mycelium pellet.
Wherein Aspergillus fumigatus described in step one is Aspergillus fumigatus YSITB I, Aspergillus fumigatus YSITB I in 2015 " cigarette is bent
Lignin research in mould YSITB I bacterial strain screening and degraded wood fibre thereof " (Yuan Junchao, Lv Bao bend honest girl etc. Agriculture In Hubei Province section
Learn .2015,54 (17)) disclosed in article.
Bacillus cereus described in step one is bacillus cereus X-10-1-2.Described bacillus cereus X-10-
1-2 in 2006 " researchs of two strain bacillus cellulase-producings " (Yan Hong, Yang Qian, Wang Xiguo. chemistry of forest product and work
Industry .2006,26 (2)) disclosed in article.
Detailed description of the invention two: present embodiment is unlike detailed description of the invention one: Aspergillus fumigatus spore in step one
The concentration of fullness over the chest during pregnancy liquid is 3 × 106Individual/mL～7 × 106Individual/mL.Other is identical with detailed description of the invention one.
Detailed description of the invention three: present embodiment is unlike detailed description of the invention one: Aspergillus fumigatus spore in step one
The concentration of fullness over the chest during pregnancy liquid is 5 × 106Individual/mL.Other is identical with detailed description of the invention one.
Detailed description of the invention four: present embodiment is unlike one of detailed description of the invention one to three: wax in step one
The concentration of sample bacillus cereus bacteria suspension is 2 × 106Individual/mL～4 × 106Individual/mL.One of other and detailed description of the invention one to three
Identical.
Detailed description of the invention five: present embodiment is unlike one of detailed description of the invention one to three: wax in step one
The concentration of sample bacillus cereus bacteria suspension is 3 × 106Individual/mL.Other is identical with one of detailed description of the invention one to three.
Detailed description of the invention six: present embodiment is unlike one of detailed description of the invention one to five: cigarette in step 2
Aspergillosis spore suspension is 1:(1～8 with the volume ratio of bacillus cereus bacteria suspension).Other is with detailed description of the invention one to five
One of identical.
Detailed description of the invention seven: present embodiment is unlike one of detailed description of the invention one to five: cigarette in step 2
Aspergillosis spore suspension is 1:(3～5 with the volume ratio of bacillus cereus bacteria suspension).Other is with detailed description of the invention one to five
One of identical.
Detailed description of the invention eight: present embodiment is unlike one of detailed description of the invention one to seven: step 2 transfer
Speed is 160r/min.Other is identical with one of detailed description of the invention one to seven.
Detailed description of the invention nine: present embodiment is unlike one of detailed description of the invention one to eight: in step 2
Temperature is that in the shaking table of 28 DEG C, 3d is cultivated in concussion.Other is identical with one of detailed description of the invention one to eight.
Detailed description of the invention ten: present embodiment is unlike one of detailed description of the invention 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.Other is identical with one of detailed description of the invention one to nine.
Detailed description of the invention 11: present embodiment co-immobilization mycelium pellet is used for lignin degrading, cellulose, half fiber
Element, dyestuff and heavy metal ion, described dyestuff is Congo red, peacock blue or crystal violet.
Elaborating embodiments of the invention below, following example are entered under premised on technical solution of the present invention
Row is implemented, and gives detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following reality
Execute 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 and is 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, adding distilled water and be made into 1000mL, pH value is natural, 120 DEG C of sterilizing 20min.This culture medium
For preserving 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 inoculating loop
Under, it is linked in sterilized water, making concentration is 1 × 106The spore suspension of individual/mL (counting method of blood cell mensuration), 4 DEG C of Refrigerator stores
Standby.
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 in sterilized water, and making concentration is 5 × 106The spore suspension of individual/mL (counting method of blood cell mensuration), 4 DEG C
Refrigerator store is standby.
Wherein Aspergillus fumigatus described in step one is Aspergillus fumigatus YSITB I, Aspergillus fumigatus YSITB I in 2015 " cigarette is bent
Lignin research in mould YSITB I bacterial strain screening and degraded wood fibre thereof " (Yuan Junchao, Lv Bao bend honest girl etc. Agriculture In Hubei Province section
Learn .2015,54 (17)) disclosed in article.
Bacillus cereus described in step one is bacillus cereus X-10-1-2.Bacillus cereus X-10-1-2 is
In 2006 " researchs of two strain bacillus cellulase-producings " (Yan Hong, Yang Qian, Wang Xiguo. chemistry of forest product with industry
.2006,26 (2)) disclosed in article.
Two, the preparation method of mycelium pellet:
Fungi pellet: take 5mL Aspergillus fumigatus bacteria suspension, is linked into the 250mL cone containing 100mL mycelium pellet balling-up culture fluid
In shape bottle, being 160r/min in rotating speed, temperature is that in the shaking table of 30 DEG C, 3d is cultivated in concussion.
Compound bacteria mycelium pellet: take 5mL Aspergillus fumigatus bacteria suspension and 20mL bacillus cereus bacteria suspension, be linked into together and contain
In the 250mL conical flask of 100mL mycelium pellet balling-up culture fluid, being 160r/min in rotating speed, temperature is to shake in the shaking table of 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 Electronic Speculum figure is as shown in figure 11.
Aspergillus fumigatus and bacillus cereus add in mycelium pellet balling-up culture fluid after fermentation, and in compound bacteria-fermented liquid, strain density is in time
The curve of change is as shown in figure 12.As seen from Figure 12, prolongation over time, the bacterium amount in fermentation liquid constantly declines, former
Cause is that the equal appendix of thalline suffers to mycelium pellet, so bacterium amount constantly declines.
Three, the mycelium pellet degraded to paper waste
Paper waste culture medium is by 100g Caulis et Folium Oryzae (oven-dry weight), 20gNaOH, 0.5g anthraquinone, 0.795g sodium sulfide and 580mL
Distilled water forms, and is warming up to 125 DEG C, is incubated 2h.
By paper waste culture medium with the dilution proportion of 1:4, pipette in the conical flask that 100mL joins 250mL, after sterilizing
Add the wet mycelium pellet of 5g, be placed in isothermal vibration incubator, 28 DEG C, cultivate under conditions of 160r/min, to be not added with at mycelium pellet
The culture medium of reason is blank, processes under the same conditions, timing sampling, measures the content of lignin in solution.Described wet
Mycelium pellet is the fungi pellet in step 2 or compound bacteria mycelium pellet.
The insoluble lignin of acid: filtered by solution, the sulfuric acid solution with 3% regulates pH to 3-4, dries precipitum to permanent
Weight, is the content of sour insoluble lignin in solution.
The molten lignin of acid: the sulfuric acid solution with 3% regulates pH to 3-4, stands overnight, measures in supernatant under 205nm
The content of lignin.
Wherein in solution, the quality of lignin is sour insoluble lignin and acid molten lignin sum.
Four, cellulose and the mensuration of hemicellulose
Take 0.1g lignin sample, add 5mL acetic acid-nitric acid mixed liquor, boiling water bath heats 20min, cooled and filtered.
Take 1mL filtrate, add 4mL orcinol reagent, 100 DEG C of insulation 15min, at 660nm, survey OD value.Obtained by xylose standard curve
Sugar amount, is multiplied by coefficient 0.9 and is the content of hemicellulose.Filtering residue washing with acetone twice, 60 DEG C are dried to constant weight, are placed in beaker
Middle addition 5mL72% sulphuric acid, 20 DEG C of hydrolysis 3h, add water 45mL, ambient temperature overnight, and next day filters, and takes 2mL filtrate and adds 5mL anthrone
Reagent, 100 DEG C of insulation 10min, survey OD value at 620nm, glucose standard curve obtain sugar amount, be multiplied by coefficient 0.9 and be
The content of cellulose.
Degradation rate calculates: before degradation rate (DR)=(inoculation before certain component content-inoculation after certain component content)/inoculation certain
Component content × 100%
Acetic acid described in step 4-nitric acid mixed liquor is by mixing that 10mL concentrated nitric acid and 100mL80% acetic acid (v/v) form
Close liquid.
Five, the mycelium pellet mensuration to dye adsorption ability
Dyestuff culture fluid includes congo red culture medium, malachite green oxalate dyestuff culture medium and Crystal Violet Dye culture medium,
Congo red culture medium: Congo red 100mg, adds distilled water and is made into 1000mL, and pH value is natural, 120 DEG C of sterilizings
20min。
Malachite green oxalate dyestuff culture medium: malachite green oxalate 100mg, adds distilled water and is made into 1000mL, and pH value is natural, and 120 DEG C go out
Bacterium 20min.
Crystal Violet Dye culture medium: crystal violet 100mg, adds distilled water and is made into 1000mL, and pH value is natural, 120 DEG C of sterilizings
20min。
Taking the wet mycelium pellet of 5g, join in the 250mL conical flask of the dyestuff culture fluid containing 100mL, the concentration of dyestuff is
100mg/L, the kind of dyestuff is Congo red, malachite green oxalate, crystal violet.It is placed in isothermal vibration incubator, 28 DEG C, 160r/min
Under conditions of cultivate.Timing sampling, takes the supernatant after centrifugal filtration, in maximum absorption wave strong point UV, visible light spectrophotometric
Measure its absorbance A1, not inoculate the absorbance A of the dyestuff culture medium of mycelium pellet0For comparison, calculate percent of decolourization.The wherein the Congo
Maximum absorption wavelength red, malachite green oxalate, crystal violet is respectively 506nm, 617.4nm and 586nm.Described wet mycelium pellet is step
Fungi pellet in two or compound bacteria mycelium pellet.
The computing formula of percent of decolourization is as follows:
Percent of decolourization (%)=[(A0-A1)/A1] × 100%
Experimental result is as follows:
1, single fungi pellet and compound bacteria mycelium pellet process the Performance comparision of various wastewater
Paper waste culture medium is by 100g Caulis et Folium Oryzae (oven-dry weight), 20gNaOH, 0.5g anthraquinone, 0.795g sodium sulfide and 580mL
Distilled water forms, and is warming up to 125 DEG C, is incubated 2h
5g fungi pellet and 5g compound bacteria mycelium pellet are inoculated in paper waste culture medium respectively, heavy metal ion is cultivated
In base and dyestuff culture medium, the liquid amount of three kinds of culture medium is 100mL (250mL conical flask), is 28 DEG C in temperature, and rotating speed is
In the shaking table of 160r/min, 3d, sampling are cultivated in concussion, measure two kinds of mycelium pellets to the degradation rate of paper waste, heavy metal ion
Adsorption rate and percent of decolourization to dyestuff.Two kinds of mycelium pellets are shown in Table 1 to the result of waste water.
Table 1 fungi pellet and compound bacteria mycelium pellet process the Performance comparision of various waste water
| 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 degraded of paper waste, fungi pellet is to lignin, cellulose, hemicellulose
Degradation rate is respectively 34.05%, and 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
Ball is compared with fungi pellet for the degraded of lignin, and degradation rate exceeds more than 10%.For the absorption of heavy metal ion, fungus 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
It is respectively 65.96% and 52.33%.The absorption of compound bacteria mycelium pellet more single fungi pellet heavy metal ion, adsorption rate
Improve about 15%.For the process of waste water from dyestuff, Congo red, percent of decolourization peacock blue, crystal violet are divided by fungi pellet
It is not 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 fungi pellet more than 20%.
Cultivate it addition, bacillus cereus X10-1-2 is inoculated into Testa Tritici with compound bacteria mycelium pellet by identical inoculum concentration
In base and Caulis et Folium Oryzae culture medium, in 30 DEG C, rotating speed be 160r/min shaking table in concussion cultivate 5d, measure bacillus cereus X10-
1-2 and compound bacteria mycelium pellet, to the degradation rate of cellulose, hemicellulose, lignin in Testa Tritici and Caulis et Folium Oryzae, the results are shown in Table 2.
Wheat bran medium is by 3.0g Na2HPO4、2.0g(NH4)2SO4, 0.5g carbamide, 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.
Caulis et Folium Oryzae culture medium is by 3.0g Na2HPO4、2.0g(NH4)2SO4, 0.5g carbamide, 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% Caulis et Folium Oryzae,
0.1% peptone, 1% yeast powder, adjusting pH value is 7.0～7.2.
Table 2 bacillus cereus X10-1-2 and compound bacteria mycelium pellet process the Performance comparision of various waste water
From data above, the performance of compound bacteria mycelium pellet is superior to single fungi pellet, simultaneously also superior to single
Bacillus cereus X10-1-2.Compound bacteria mycelium pellet is a kind of two microorganisms immobilization system, contains lignin degrading simultaneously
Fungus and the antibacterial of degraded cellulose, two strains have the performance of complementation, serve the effect mutually promoted, both had lignin
Degradation capability has again cellulose degradation vigor.
2, compound bacteria mycelium pellet processes the performance study of various waste water
1. paper waste is processed
The performance of compound bacteria mycelium pellet degraded paper waste under different temperatures:
5g compound bacteria mycelium pellet is inoculated in the 250mL conical flask containing 100mL paper waste culture medium, in rotating speed is
160r/min, temperature is respectively in the shaking table of 24 DEG C, 28 DEG C, 30 DEG C, 34 DEG C, 37 DEG C and 40 DEG C concussion and cultivates, and samples every 2d,
Measure the degradation rate of lignocellulose.Record the maximum that ligocellulose degradation at each temperature leads and see Fig. 1, in figure ◆ represent wood
The degradation rate of quality, ■ represents 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 the highest, up to 58.01%.When 30 DEG C and 34 DEG C, the degradation rate of cellulose and hemicellulose reaches the highest, respectively
It is 45.39% and 57.45%.When temperature is 40 DEG C, the degradation rate of hemicellulose remains to reach 37.91%, illustrates at high temperature
In the environment of, mycelium pellet still has well degraded to paper waste.
The performance of compound bacteria mycelium pellet degraded paper waste under different pH:
5g compound bacteria mycelium pellet is inoculated in the 250mL conical flask containing 100mL paper waste culture medium, regulates culture medium
PH be respectively 2,3,5,7,9,11 and 12, be 160r/min in rotating speed, temperature is that in the shaking table of 28 DEG C, concussion is cultivated, every 2d
Sampling, measures the degradation rate of lignocellulose, records the maximum that under each pH value, ligocellulose degradation leads and see Fig. 2, in figure ◆
Representing the degradation rate of lignin, ■ represents 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 suppress
The generation of lignin-degrading enzymes, is unfavorable for the degraded of lignin in papermaking wastewater.When pH is 5, Lignin degradation rate is the highest, up to
To 61.38%.Along with the increase of pH, Lignin degradation rate decreases, and pH is under the system of 9, and the degradation rate of lignin remains to
Reaching 31.47%, cellulose and hemicellulose degradation rate all can maintain more than 40% under the system of 5～11, illustrate partially
In the environment of acidity and alkalescence, papermaking wastewater be can still provide for well degrading by compound bacteria mycelium pellet.
The performance of compound bacteria mycelium pellet degraded paper waste under different time:
5g compound bacteria mycelium pellet is inoculated in the 250mL conical flask containing 100mL paper waste culture medium, in rotating speed is
160r/min, temperature is that in the shaking table of 28 DEG C, concussion is cultivated, and every day samples, and measures the degradation rate of lignocellulose, and result is shown in figure
3, in figure ◆ representing the degradation rate of lignin, ■ represents the degradation rate of cellulose, the degradation rate of × expression hemicellulose.
As seen from Figure 3, along with the prolongation of incubation time, the degradation rate of lignin is presented first to raise and drops afterwards by mycelium pellet
Low trend, this is because what lignin was mainly made up of sour molten lignin and the insoluble lignin of acid, big in system during beginning
Molecular wood quality fragment gradually decreases, and degradation rate gradually rises, and when cultivating 144h, degradation rate reaches to be up to 63.61%.
But, in degradation process, the macromolecular gluco in sour molten lignin can be degraded to small molecule segment by mycelium pellet, and therefore acid is not
The content of molten lignin can increase, and the content of thoroughly removing of lignin reduces, and total Lignin degradation rate reduces.Due to mycelium pellet
Having the strongest degradation capability, along with the degraded of little molecular wood quality fragment, Lignin degradation rate gradually rises again.Compound bacteria bacterium
Pompon also has the highest degradation capability to cellulose and hemicellulose, and when 72h and 60h, high degradation rate is respectively
49.05% and 63.36%.
Mycelium pellet recycling process paper waste:
Investigate stability and the recycling effect of compound bacteria mycelium pellet, 5g compound bacteria mycelium pellet is inoculated in containing 100mL
In the 250mL conical flask of paper waste culture medium, being 160r/min in rotating speed, temperature is that in the shaking table of 28 DEG C, 5d is cultivated in concussion,
After completing to circulate for the first time, this compound bacteria mycelium pellet is linked in new wastewater medium, processes 5d at identical conditions.
So circulation 4 times, the results are shown in Table 3.
Table 3 compound bacteria mycelium pellet recycling processes paper waste result
As shown in Table 2, in primary circulation, mycelium pellet is fine to the degradation effect of lignocellulose, lignin, fibre
Dimension element, the degradation rate of hemicellulose are respectively 51.27%, 41.58%, 57.1%, in second time to the circulation of third time, and bacterium
The degradation rate of lignocellulose has been declined by pompon, and it is rough that mycelium pellet surface becomes, and spheroid color becomes dark-brown, but right
The degradation rate of three kinds of lignocellulose remains to reach more than 20%.The when of until being recycled to the 4th time, mycelium pellet just starts
The phenomenon of existing self-dissolving.This shows that mycelium pellet can maintain activity for a long time, it is possible to lasting process waste water.
2. the process of compound bacteria mycelium pellet heavy metal ion waste water
The performance of compound bacteria mycelium pellet Adsorption of Heavy Metal Ions 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 and is made into 1000mL, and pH value is natural, 120 DEG C of sterilizing 20min.
Pb2+Culture medium: copper sulfate 195.32mg, adds distilled water and is made into 1000mL, and pH value is natural, 120 DEG C of sterilizing 20min.
5g compound bacteria mycelium pellet is inoculated in the 250mL conical flask containing 100mL heavy metal ion culture medium, in rotating speed is
160r/min, temperature is respectively in the shaking table of 20 DEG C, 24 DEG C, 28 DEG C, 30 DEG C, 34 DEG C, 37 DEG C and 40 DEG C concussion and cultivates, every 1d
Sampling, measures heavy metal ion adsorbed rate.The maximum recording the most heavy metal ion adsorbed rate is shown in Fig. 4, in figure ◆ table
Show that Cu, ■ represent Pb.
As shown in Figure 4, the absorption of the temperature heavy metal ion in the range of investigation has a certain impact.At 28 DEG C～34 DEG C
In the range of adsorption effect all fine, compound bacteria mycelium pellet is to Cu2+Adsorption rate reach the highest, to Pb when 28 DEG C2+Absorption
Rate reaches the highest when 30 DEG C, 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, and the absorption that all can affect compound bacteria mycelium pellet heavy metal ion too high or too low for temperature is described.
The performance of compound bacteria mycelium pellet Adsorption of Heavy Metal Ions under different pH:
Being inoculated in by 5g compound bacteria mycelium pellet in the 250mL conical flask containing 100mL heavy metal ion culture medium, regulation is cultivated
The pH of base is respectively 2,3,5,7,9,11 and 12, is 160r/min in rotating speed, and temperature is that in the shaking table of 28 DEG C, concussion is cultivated, every
1d samples, and measures heavy metal ion adsorbed rate.Record the maximum of heavy metal ion adsorbed rate under each pH value and see Fig. 5, in figure ◆
Represent that Cu, ■ represent Pb.
The pH value of solution can affect metal ion and the chemical state of mycelium pellet cell surface simultaneously, and research pH value is to mycelia
The impact tool of ball Adsorption of Heavy Metal Ions is of great significance.As shown in Figure 5, when pH value is less than 5, compound bacteria mycelium pellet
To Cu2+And Pb2+Adsorption rate the lowest, this is because when pH is less, proton occupies the major part absorption on mycelium pellet surface
Site, makes the adsorption efficiency of heavy metal ion reduce.PH value adsorption effect in the range of 5～9 is all fine, when pH value is 5
Time, Cu2+And Pb2+Adsorption rate reach maximum, respectively 73.21% and 59.29%.When pH value is more than 9, the OH in solution-
Ion with metal ion generation Competition, can cause adsorption rate to reduce with the functional groups on mycelium pellet surface then.Knot
Fruit shows that compound bacteria mycelium pellet heavy metal ion under meta-acid and alkaline environment all has good adsorption effect.
The performance of compound bacteria mycelium pellet Adsorption of Heavy Metal Ions under different time:
5g compound bacteria mycelium pellet is inoculated in the 250mL conical flask containing 100mL heavy metal ion culture medium, in rotating speed is
160r/min, temperature is that in the shaking table of 28 DEG C, concussion is cultivated.When starting to measure, sample every 30min, measure heavy metal ion
Adsorption rate, result is shown in Fig. 6, in figure ◆ represent that Cu, ■ represent Pb.
It will be appreciated from fig. 6 that compound bacteria mycelium pellet is to Cu2+And Pb2+Absorption just respectively reached in 10 hours
63.99% and 37.53%, at short notice in solution the lowering of concentration of heavy metal ion quickly, this is because this absorption phase
For surface quick adsorption stage, the various active groups on mycelium pellet cell wall and the absorption of heavy metal ion ligand complex.With
After adsorption time in, the concentration of heavy metal ion in solution slowly declines until adsorption equilibrium, this is because along with adsorbance
Increase, the absorption of mycelium pellet cell wall heavy metal ion has reached saturated, and free heavy metal ion is thin to enter into
Intracellular, suffered resistance will increase, and therefore reaching to adsorb the saturated time will be the longest.When 48h and 72h, compound bacteria bacterium
Pompon is to Cu2+And Pb2+Adsorption rate reach maximum, respectively 76.96% and 61.32%.
3. the process to multiple waste water from dyestuff
The performance of compound bacteria mycelium pellet absorbing dye under different temperatures:
Dyestuff culture fluid includes congo red culture medium, malachite green oxalate dyestuff culture medium and Crystal Violet Dye culture medium,
Congo red culture medium: Congo red 100mg, adds distilled water and is made into 1000mL, and pH value is natural, 120 DEG C of sterilizings
20min。
Malachite green oxalate dyestuff culture medium: malachite green oxalate 100mg, adds distilled water and is made into 1000mL, and pH value is natural, and 120 DEG C go out
Bacterium 20min.
Crystal Violet Dye culture medium: crystal violet 100mg, adds distilled water and is made into 1000mL, and pH value is natural, 120 DEG C of sterilizings
20min。
5g compound bacteria mycelium pellet is inoculated in the 250mL conical flask containing 100mL dyestuff culture medium, is 160r/ in rotating speed
Min, temperature is respectively in the shaking table of 20 DEG C, 24 DEG C, 28 DEG C, 30 DEG C, 34 DEG C and 40 DEG C concussion and cultivates, and samples every 12h, measures
The percent of decolourization of dyestuff, the maximum recording the most dye decolored rate is shown in Fig. 7, in figure ◆ representing Congo red, ■ represents peafowl
Indigo plant, ▲ represent crystal violet.
As shown in Figure 7, compound bacteria mycelium pellet absorbing dye is had a great impact by temperature, when temperature is at 20 DEG C～40 DEG C
Time, percent of decolourization has the trend of a first increases and then decreases along with the increase of temperature.Within the temperature range of 28 DEG C～34 DEG C, multiple
Close bacterium mycelium pellet the most fine to the decolorizing effect of three kinds of dyestuffs.When temperature reaches 28 DEG C, compound bacteria mycelium pellet to peacock blue and
The adsorption effect of Congo red is best, and percent of decolourization is up to 94.75% and 96.43%；When temperature 30 DEG C, 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 is to three kinds of dyes
The percent of decolourization of material is all about 30%.Thus it is inferred that the change of temperature can affect the secretion of lignocellulolyticenzymes and change
Becoming structure and the character of enzyme, temperature is too high can reduce the compound bacteria mycelium pellet percent of decolourization to dyestuff.
The performance of compound bacteria mycelium pellet absorbing dye under different pH:
Being inoculated in by 5g compound bacteria mycelium pellet in 100mL (250mL conical flask) dyestuff culture medium, the pH of regulation culture medium divides
Not being 2,3,5,7,9,11 and 12, be 160r/min in rotating speed, temperature is that in the shaking table of 28 DEG C, concussion is cultivated, and samples every 12h,
Measure the percent of decolourization of dyestuff, record the maximum of dye decolored rate under each PH and see Fig. 8, in figure ◆ representing Congo red, ■ represents hole
Passeris montani saturati is blue, ▲ represent crystal violet.
Dyestuff is decoloured and mainly relies on degraded the modes such as the catalysis oxidation of enzyme system, reduction to break by compound bacteria mycelium pellet
The unsaturated conjugated key of bad dyestuff and color development system, enzyme's reaction speeding is had a great impact by pH, only in Optimal pH condition
Under, enzyme just can show best catalysis activity.As shown in Figure 8, pH is in the range of 5～9, and compound bacteria mycelium pellet is to three kinds of dyes
The percent of decolourization of material is all up to more than 70%.When pH is 5, compound bacteria mycelium pellet is to Congo red and peacock blue decolorizing effect
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%.Result shows that compound bacteria mycelium pellet all has well absorption in the environment of inclined acid and alkaline to dyestuff
Effect.
The performance of compound bacteria mycelium pellet absorbing dye under different time:
5g compound bacteria mycelium pellet is inoculated in the 250mL conical flask containing 100mL dyestuff culture medium, is 160r/ in rotating speed
Min, temperature is that in the shaking table of 28 DEG C, concussion is cultivated, and when starting to measure, samples every 30min, measures the percent of decolourization of dyestuff, result
See Fig. 9, in figure ◆ representing Congo red, ■ represents peacock blue, ▲ represent crystal violet.
As shown in Figure 9, the adsorbance of three kinds of dyestuffs is increased the most fast within initial a period of time by compound bacteria mycelium pellet
Speed, the most gradually slows down until adsorption equilibrium.For Congo red, peacock blue and crystal violet these three dyestuff, within initial 12h
Percent of decolourization just can reach more than 70%, decolorization rate is higher.When 6h, compound bacteria mycelium pellet is to the percent of decolourization of Congo red
Height, can reach 97.92%.When cultivating 48h, compound bacteria mycelium pellet is to peacock blue, and the adsorption effect of crystal violet is best, the highest de-
Color rate is respectively 95.61% and 88.63%.Result shows that the decolouring of three kinds of dyestuffs is all achieved well by compound bacteria mycelium pellet
Effect.
Claims (10)
1. a preparation method with the co-immobilization mycelium pellet removing multiple pollutant function, it is characterised in that the method bag
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 inoculating loop
Under, it is linked in sterilized water, making concentration is 1 × 106Individual/mL～9 × 106The Aspergillus fumigatus spores suspension of individual/mL；
Bacillus cereus bacteria suspension: taken out by the bacillus cereus solid slope being preserved under 4 DEG C of environment, will with inoculating loop
It scrapes, and is linked in sterilized water, and making concentration is 1 × 106Individual/mL～5 × 106The bacillus cereus bacteria suspension of individual/mL；
Two, the preparation method of compound bacteria mycelium pellet:
Take Aspergillus fumigatus spores suspension and the bacillus cereus bacteria suspension of step one preparation of step one preparation, be linked into together
In 100mL mycelium pellet balling-up culture medium, being 120～200r/min in rotating speed, temperature is to shake cultivation 2 in the shaking table of 24～37 DEG C
～5d, i.e. obtain compound bacteria mycelium pellet；
Wherein Aspergillus fumigatus described in step one is Aspergillus fumigatus YSITB I,
Bacillus cereus described in step one is bacillus cereus X-10-1-2.
A kind of preparation side with the co-immobilization mycelium pellet removing multiple pollutant function the most according to claim 1
Method, it is characterised in that in step one, the concentration of Aspergillus fumigatus spores suspension is 3 × 106Individual/mL～7 × 106Individual/mL.
A kind of preparation side with the co-immobilization mycelium pellet removing multiple pollutant function the most according to claim 1
Method, it is characterised in that in step one, the concentration of bacillus cereus bacteria suspension is 2 × 106Individual/mL～4 × 106Individual/mL.
A kind of preparation side with the co-immobilization mycelium pellet removing multiple pollutant function the most according to claim 1
Method, it is characterised in that in step 2, Aspergillus fumigatus spores suspension is 1:(1～8 with the volume ratio of bacillus cereus bacteria suspension).
A kind of preparation side with the co-immobilization mycelium pellet removing multiple pollutant function the most according to claim 1
Method, it is characterised in that in step 2, Aspergillus fumigatus spores suspension is 1:(3～5 with the volume ratio of bacillus cereus bacteria suspension).
A kind of preparation side with the co-immobilization mycelium pellet removing multiple pollutant function the most according to claim 1
Method, it is characterised in that step 2 medium speed is 160r/min.
A kind of preparation side with the co-immobilization mycelium pellet removing multiple pollutant function the most according to claim 1
Method, it is characterised in that in step 2,3d is cultivated in concussion in the shaking table that temperature is 28 DEG C.
A kind of preparation side with the co-immobilization mycelium pellet removing multiple pollutant function the most 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/
LKH2PO4、2g/L MgSO4.7H2O and distilled water are made, and pH value is adjusted to 5,120 DEG C of sterilizing 20min.
9. the application of the co-immobilization mycelium pellet that prepared by the method for claim 1, it is characterised in that co-immobilization mycelia
Ball is for lignin degrading, cellulose, hemicellulose, dyestuff and heavy metal ion.
Application the most according to claim 9, it is characterised in that described dyestuff is Congo red, peacock blue or crystal violet.
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