CN102296058A - Immobilization operating method of white rot fungi - Google Patents
Immobilization operating method of white rot fungi Download PDFInfo
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- CN102296058A CN102296058A CN 201110203842 CN201110203842A CN102296058A CN 102296058 A CN102296058 A CN 102296058A CN 201110203842 CN201110203842 CN 201110203842 CN 201110203842 A CN201110203842 A CN 201110203842A CN 102296058 A CN102296058 A CN 102296058A
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- rot fungi
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- wood chip
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Abstract
The invention discloses an immobilization operating method of white rot fungi, comprising the following steps of: firstly inoculating a strain into a conical flask with 150ml of a liquid medium on an aseptic operating board, adjusting the pH value of the liquid medium to 5.0, and culturing for 2 days in a constant temperature water tank of 30 DEG C; weighing 10g of peanut shells, placing the peanut shells into a H2O2 solution at the concentration of 2% to immerse for 30 minutes, washing to neutral by the use of distilled water, adding the liquid medium cultured for 2 days, keeping culturing for 12-24 hours, filtering out the culture medium to obtain the white rot fungi which has undergone wood chip adsorption and immobilization. The method provided by the invention has advantages of simple preparation, cost low, persistence as well as stable and high degradation efficiency.
Description
Technical field
The present invention relates to a kind of white-rot fungi be carried out the immobilization method of operating.
Background technology
Immobilization technology comprises enzyme immobilization technology and immobilized cell technology.Immobilized cell technology is to develop from enzyme immobilization technology gradually since the seventies, it be meant by the method for physics or chemistry will disperse, the free microorganism cells is fixed in a certain restriceted envelope zone, to improve the concentration of microorganism cells, make it keep higher biological activity and the method for recycling.Because this method does not need enzyme is extracted from cell, and need not purifying, thereby the enzyme activity loss is little.As seen, immobilized cell technology has its unique gesture than enzyme immobilization technology, and this also makes immobilized cell technology be developed rapidly and be perfect.
Immobilization technology has been widely used in the Industrial Wastewater Treatment because of characteristics such as its distinctive cell density height, toxin immunity are strong and can recycle, and effect is better, and is wherein more with absorption method and entrapping method research, uses also wider.Adsorption of immobilization is the electrostatic interaction that utilizes between carrier and the microorganism, makes microorganism be adsorbed on carrier surface and is fixed, and the selection of carrier is particularly crucial.Research at present mainly contains mineral-type, polysaccharide and protein-based, natural polymer gel-like and organic synthesis high-molecular gel class carrier.Existing scholar utilizes some natural organic carrier immobilizing white rot fungi degradation of phenol waste water and other waste water, but less for the research of carrier selection aspect.Become emphasis of the present invention.
Summary of the invention
The object of the present invention is to provide and a kind of white-rot fungi has been carried out the immobilization method of operating.The present invention has preparation simply, and cost is low, and lasting and firm, degradation efficiency is firmly efficient, the characteristics of the treatment of wastes with processes of wastes against one another.
In order to achieve the above object, technical scheme of the present invention is:
A kind of white-rot fungi is carried out the immobilization method of operating, comprise the steps: at first bacterial classification to be inoculated in the Erlenmeyer flask that the 150ml liquid nutrient medium is housed in the aseptic technique platform, regulating liquid medium pH value is 5.0, cultivates 2d in 30 ℃ of waters bath with thermostatic control; Take by weighing the 10g Pericarppium arachidis hypogaeae, put into 2% H
2O
2Soak 30min in the solution, be washed till neutrality, add and to have cultivated in the liquid nutrient medium of 2d with distilled water, continue to cultivate 12-24h after the time elimination nutrient solution promptly get the white-rot fungi of wood chip adsorption of immobilization.
Described Pericarppium arachidis hypogaeae is by a kind of replacement in straw, wood chip, corn cob, sugarcane, the gac.
Described liquid culture based formulas is: KH
2PO
41. 0 g/L, NaH
2PO
40. 2 g/L, MgSO
47 H
2O 0. 5 g/L, VB1 0. 1mg/L, CaCl
20. 1mg/L, FeSO
47 H
2O 0. 1mg/L, ZnSO
47 H
2O 0. 01mg/L, CuSO
45 H
2O 0. 2mg/L, glucose 1. 0 g/L, ammonium tartrate 0.11 g/L, the pH value is 5.0.
Beneficial effect of the present invention is: the present invention has selected several agricultural waste products as fixation support, and the percent of decolourization of taking the immobilized white-rot fungi simulated wastewater of absorption method has been carried out preliminary study.The immobilized white-rot fungi of method of the present invention has preparation simply, and cost is low, and lasting and firm, degradation efficiency is firmly efficient, the characteristics of the treatment of wastes with processes of wastes against one another.
Description of drawings
Fig. 1 is that embodiment of the invention blank assay ultraviolet spectrophotometer is measured figure;
Fig. 2 is that the immobilized white-rot fungi ultraviolet spectrophotometer of embodiment of the invention Pericarppium arachidis hypogaeae is measured figure;
Fig. 3 is that the immobilized white-rot fungi ultraviolet spectrophotometer of embodiment of the invention wood chip is measured figure;
Fig. 4 is that the immobilized white-rot fungi ultraviolet spectrophotometer of embodiment of the invention straw is measured figure;
Fig. 5 is that the corn core immobilized white-rot fungi ultraviolet spectrophotometer of the embodiment of the invention is measured figure;
Fig. 6 is that the immobilized white-rot fungi ultraviolet spectrophotometer of embodiment of the invention gac is measured figure;
Fig. 7 is that the immobilized white-rot fungi ultraviolet spectrophotometer of embodiment of the invention sugarcane is measured figure;
Fig. 8 is all immobilizing white rot fungi decolorizing effect comparison diagrams of the embodiment of the invention.
Embodiment
Embodiment 1
The collection of present embodiment white-rot fungi is from the rotten mould wood of white, separate white hypha, be inoculated on the white-rot fungi primary dcreening operation nutritive medium after the sterilization, put in 39 ℃ the incubator and cultivated through 3 days, after waiting to grow bacterium colony, through the row separation and purification, the white-rot fungi that is inoculated into after the sterilization sieves on the substratum again, judges that by forming the methyl catechol variable color and forming chromosphere white bacterial classification is a white-rot fungi.Present embodiment is taked to cultivate the mode of afterwards adsorbing earlier, the fixing cultivation in basic medium, and the liquid culture based formulas of preparation is as follows: KH
2PO
41. 0 g/L, NaH
2PO
40. 2 g/L, MgSO
47 H
2O 0. 5 g/L, VB1 0. 1mg/L, CaCl
20. 1mg/L, FeSO
47 H
2O 0. 1mg/L, ZnSO
47 H
2O 0. 01mg/L, CuSO
45 H
2O 0. 2mg/L, glucose 1. 0 g/L, ammonium tartrate 0.11 g/L, agar 18 g/L, the pH value is 5.0.
The bacterial classification of solid culture is inoculated in the Erlenmeyer flask that the 150ml nutrient solution is housed according to the working specification of strictness in the aseptic technique platform, regulating liquid medium pH value is 5.0, cultivates 2d in 30 ℃ of waters bath with thermostatic control.Take by weighing the 10g Pericarppium arachidis hypogaeae, put into 2% H
2O
2Soak 30min in the solution, be washed to neutrality with heating up in a steamer, add and cultivated in the liquid nutrient medium of 2d, the elimination nutrient solution promptly gets the white-rot fungi of wood chip adsorption of immobilization after continuation cultivation for some time.Other are also operation as stated above of material in 5, obtains the white-rot fungi of the adsorption of immobilization of straw, wood chip, corn cob, sugarcane, gac.Will be by the white-rot fungi of various material adsorption of immobilization, every kind is divided into two parts, and every part of 5g is placed into respectively in the aseptic Erlenmeyer flask that 150ml distilled water is housed, and 2 parts every group, has 6 groups.In addition, a blank group is set again, experimentizes.Then, add the aseptic simulated wastewater of 10ml in these 13 bottles of Erlenmeyer flasks, mix.Afterwards 13 samples are put into the simulated wastewater of degrading in 30 ℃ the constant incubator (in 1L distilled water, add Reactive blue (KN-R) 2g, disperse emerald green blue (S-GL) 1.5g, carry out uniform mixing).Adopt 752 ultraviolet-visible pectrophotometers to measure simulated wastewater and the absorbancy that is total to nutrient solution and the relation of wavelength, the result shows that its maximum absorption wavelength is positioned at the 300nm place.In 96h,, 13 samples are analysed photometry every 12 h.If initially altogether the absorbancy of nutrient solution is Ai, the residue absorbancy is Af, then the percent of decolourization of waste water=(Ai-Af)/Ai * 100%.Join the Erlenmeyer flask 2～3h of white-rot fungi of material adsorption of immobilization when simulated wastewater after, thalline becomes blue because of the absorbed portion dyestuff.It is blue that waste water from dyestuff still is.Behind about 48h, thalline and the waste water from dyestuff observed under 30 ℃ of culture condition of constant temperature transfer lighter cyan to.Behind the 96h, thalline and nutrient solution are near colourless.Waste water from dyestuff color in the blank assay does not change substantially over time.Following Fig. 1-the 8th, each material form measuring the gained data organization every 12h with ultraviolet spectrophotometer.
Experimentation is observed, join the Erlenmeyer flask 2～3h of white-rot fungi of material adsorption of immobilization when simulated wastewater after, thalline becomes blue because of the absorbed portion dyestuff.It is blue that waste water from dyestuff still is.Behind about 48h, thalline and the waste water from dyestuff observed under 30 ℃ of culture condition of constant temperature transfer lighter cyan to.Behind the 96h, thalline and nutrient solution are near colourless.Waste water from dyestuff color in the blank assay does not change substantially over time.Following Fig. 1-the 8th, each material form measuring the gained data organization every 12h with ultraviolet spectrophotometer.
As seen from Figure 1, blank group experimental simulation decolorization rate of wastewater is almost nil.
As seen from Figure 2, the immobilized white-rot fungi of Pericarppium arachidis hypogaeae is under 30 ℃ cultivation, and decolorizing effect is apparent in view, and percent of decolourization presents complicated variation phenomenon, percent of decolourization rising originally, percent of decolourization decline subsequently, rising again afterwards in time.There is a process that significantly earlier rises and afterwards descend the centre, this may be to have produced some intermediate products in the degradation process to cause, the major cause that causes this phenomenon is two parts of biochemical degradation that the process of white rot fungus degrading dyestuff comprises physical adsorption and thalline, thalline excretory xylogen peroxidase participates in reaction after the physical adsorption, form the middle mixture of highly active enzyme, dye molecule is oxidized to free radical, and then produce many different free radicals with chain reaction, impel the dye molecule oxidation, reach the purpose of biochemical degradation.Because the biochemical degradation process occurs in outside the thalline, so when white rot fungus degrading behind a part of dyestuff in the solution, dye strength in the solution descends, and causes being adsorbed on the part dyestuff that is not degraded on the thalline and separates again and be drawn onto in the solution, causes the rising of analysing photometric quantity.
As seen from Figure 3, the white-rot fungi decolorizing effect of wood chip group is more general, does not have significantly to occur as the middle process that rises and afterwards descend earlier of peanut group, and is final also lower than the peanut group at the percent of decolourization of 96h.When 70h to 96h, decoloring ability has almost arrived threshold value, and therefore, decolorizing effect is general.
Find out that by Fig. 4 the decolorizing efficiency of the immobilized white-rot fungi of straw when 23h to 38h is high especially, and after 40h, the level and smooth stage has appearred in curve.To the visual inspection of two Erlenmeyer flasks, the blueness in the simulated wastewater is almost gone out, but the color after handling becomes pale brown look.This mainly is because straw itself contains certain pigment, and soaking to make has for a long time had these pigments in the water body, caused the water body flavescence, thereby makes that analysing photometric quantity becomes than higher, has influenced the final percent of decolourization of experiment.As seen, though straw is good to the decolorizing effect of waste water, owing to reason itself can cause the variation of water body chroma, so decolorizing effect has been subjected to influence greatly.
By finding out among Fig. 5, the decolorizing effect of corn core immobilized white-rot fungi is that more stable, final percent of decolourization is also than higher on the whole.At 24h to 39h is the highest time period of decolorizing efficiency, occurred afterwards as the first process that rises and afterwards descend of that section of peanut group centre, but be not clearly, wherein can find out that small size decline is arranged for one group, its reason also is that this may be to have produced some intermediate products in the degradation process.
The immobilized white-rot fungi of gac is fine at the decolorizing effect of beginning as seen from Figure 6, and this mainly has benefited from the character of gac itself, because gac itself just has stronger adsorptivity, has the dual adsorptivity of physics and chemistry.Therefore, the initial stage, the dyestuff in the simulated wastewater is at first by charcoal absorption, and then begins degraded by the superincumbent white-rot fungi of absorption.From 40h, the immobilized white-rot fungi decolorizing efficiency of gac is just lower, and it is poor relevant that this improves decolorizing effect with gac itself to white-rot fungi.Gac is as 6 groups of a kind of inorganicss that material is unique, the white-rot fungi immobilization effect is not so good as other 5 groups agricultural organism, adding gac is that price is the highest in these materials, so cost performance is lower, the uncomfortable incompatible immobilization material of doing white-rot fungi.
The curve of sugarcane group and the curve of peanut group are more similar as seen from Figure 7, and final percent of decolourization is also than higher.At 24h-45h, during this period of time in, the immobilizing white rot fungi of sugarcane group keeps higher decolorizing effect always.Detect by an unaided eye and can find, the bagasse intrinsic colour has also become blueness, can infer to have absorbed the dyestuff in the water body.Therefore, make decolorizing effect outstanding.And a large amount of carbon, nitrogen are arranged in the bagasse, and can improve better growing conditions to white-rot fungi, improved the decolorizing efficiency of white-rot fungi.
Material with all immobilizing white rot fungis among Fig. 8 compares, and wherein effect is sugarcane and Pericarppium arachidis hypogaeae for reasonable two kinds, and percent of decolourization has reached 79% and 80.5% respectively.What decolorizing effect was relatively poor is gac and straw, respectively only 69.5% and 68%.
The decolorizing effect of corn and wood chip is general, is respectively 75% and 75.5%.
In these several carriers, straw and sugarcane material are softer, and density is less, and physical strength is poor, are difficult for settlement separate in wastewater treatment process and are easy to biological degradation, are unsuitable for the immobilization of white-rot fungi; Gac is not best as its treatment effect of inorganic carrier, and cost is the highest; Corn cob, Pericarppium arachidis hypogaeae, wood chip material are harder, and density is bigger slightly than water, and physical strength is better, and settlement separate property is good, and absorption is closely and have certain antibiont degradation capability between the microorganism.Comparatively speaking, corn cob and Pericarppium arachidis hypogaeae powder are more satisfactory carriers, and these several carrier inside all have many micro-capsules hole, and white-rot fungi is adsorbable to grow in these micro-capsules, have avoided causing because of adsorptive power is little the phenomenons of a large amount of losses of immobilized bacterium; This in addition microcapsule structure makes carrier itself have bigger specific surface area, has strengthened the adsorptive power of immobilizing white rot fungi to pollutent in the waste water.
In sum, in straw powder, Pericarppium arachidis hypogaeae, wood chip, corn cob, sugarcane, the gac, it is Pericarppium arachidis hypogaeae that the most suitable white-rot fungi carries out immobilized material, secondly is sugarcane.Have higher decoloring ability as the immobilized white-rot fungi of Pericarppium arachidis hypogaeae, and the physical strength of Pericarppium arachidis hypogaeae is good, settlement separate property is good, and bigger specific surface area is arranged, and has strengthened the percent of decolourization of immobilizing white rot fungi to simulated wastewater.
Claims (3)
1. one kind is carried out the immobilization method of operating to white-rot fungi, it is characterized in that comprising the steps: at first bacterial classification in the aseptic technique platform, be inoculated in the Erlenmeyer flask that the 150ml liquid nutrient medium is housed, regulating liquid medium pH value is 5.0, cultivates 2d in 30 ℃ of waters bath with thermostatic control; Take by weighing the 10g Pericarppium arachidis hypogaeae, put into 2% H
2O
2Soak 30min in the solution, be washed till neutrality, add and to have cultivated in the liquid nutrient medium of 2d with distilled water, continue to cultivate 12-24h after the time elimination nutrient solution promptly get the white-rot fungi of wood chip adsorption of immobilization.
2. as claimed in claim 1ly a kind of white-rot fungi is carried out the immobilization method of operating, it is characterized in that: described Pericarppium arachidis hypogaeae is by a kind of replacement in straw, wood chip, corn cob, sugarcane, the gac.
3. as claimed in claim 1ly a kind of white-rot fungi is carried out the immobilization method of operating, it is characterized in that: described liquid culture based formulas is: KH
2PO
41. 0 g/L, NaH
2PO
40. 2 g/L, MgSO
47 H
2O 0. 5 g/L, VB1 0. 1mg/L, CaCl2 0. 1mg/L, FeSO
47 H
2O 0. 1mg/L, ZnSO
47 H
2O 0. 01mg/L, CuSO
45 H
2O 0. 2mg/L, glucose 1. 0 g/L, ammonium tartrate 0.11 g/L, the pH value is 5.0.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102824896A (en) * | 2012-08-21 | 2012-12-19 | 浙江大学 | Chestnut shell adsorption material and its preparation method and use |
| CN110477234A (en) * | 2019-08-20 | 2019-11-22 | 广东省生物工程研究所（广州甘蔗糖业研究所） | A kind of synchronous method and device thereof for being coupled fruit juice and taking away the puckery taste of fungi fermentation |
-
2011
- 2011-07-21 CN CN 201110203842 patent/CN102296058A/en active Pending
Non-Patent Citations (3)
| Title |
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| 《化学与生物工程》 20080131 谢冬瑾等 吸附法固定化白腐真菌的研究 第40页右栏第2段第1-7行，第1.2.3节第1-2行，第41页图2 1-3 第25卷, 第1期 * |
| 《哈尔滨工业大学学报》 20080131 曾永刚等 白腐真菌固定化技术及其影响因素的研究进展 第141-146页 1-3 第40卷, 第1期 * |
| 《水处理技术》 20051231 李朝霞等 固定化白腐菌对造纸废水的生物降解研究 第23页第1.3.1节 3 第31卷, 第12期 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102824896A (en) * | 2012-08-21 | 2012-12-19 | 浙江大学 | Chestnut shell adsorption material and its preparation method and use |
| CN110477234A (en) * | 2019-08-20 | 2019-11-22 | 广东省生物工程研究所（广州甘蔗糖业研究所） | A kind of synchronous method and device thereof for being coupled fruit juice and taking away the puckery taste of fungi fermentation |
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Application publication date: 20111228 |