CN1405306A - Specific bacteria preparation and method for treating chemical waste water or conventional organic waste water - Google Patents
Specific bacteria preparation and method for treating chemical waste water or conventional organic waste water Download PDFInfo
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- CN1405306A CN1405306A CN02138171.2A CN02138171A CN1405306A CN 1405306 A CN1405306 A CN 1405306A CN 02138171 A CN02138171 A CN 02138171A CN 1405306 A CN1405306 A CN 1405306A
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- 241000894006 Bacteria Species 0.000 title claims abstract description 79
- 238000000034 method Methods 0.000 title claims abstract description 10
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- 239000010815 organic waste Substances 0.000 title claims description 8
- 239000002894 chemical waste Substances 0.000 title 1
- 239000002351 wastewater Substances 0.000 claims abstract description 31
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 11
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 8
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- 239000011707 mineral Substances 0.000 claims abstract description 7
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- 239000007788 liquid Substances 0.000 claims description 15
- 235000015097 nutrients Nutrition 0.000 claims description 12
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Abstract
The invention is a method to prepare the specific bacterium agent which fuses the protoplast of white-rotten ephiphyte, aboriginal bacterium YZ1 and vintage yeast, and by the gene's reformation and conformity, structures the gene engineering bacterium. The ferment parameters: ferment temperature 33 plus and minus 2 C degree, pH value 7.0 plus and minus 1; soluble oxygen in reactor >=2mg/L; mineral salt flux 0.001-0.005V/(V.d); thalli concentration 2-10 g/L; cultivating it in the culture fluid containing C, P and N. The maximal degradation area of NJU-Fhhh1 quick-degradation extract to phenyl-dimethyl acid petrochemical waste water: qmax=0.226/h.
Description
One, technical field
The present invention relates to a kind of biotechnology and application thereof for preparing the special efficacy microbiobacterial agent.
Two, background technology:
Existing in the world multiple processing waste water specific bacteria, but there are not preparation and application protoplastis to merge the patent that microbial inoculum is handled waste water.The EM of typical as Japan, the high intelligent microbial preparation HIMP of U.S. PARADISF company (High-Intelligence Microorganism Preparation) and for example, 1 liter/24 yuan.Because cost and price are too high, domestic manufacturer is generally without the processing waste water specific bacteria of import, and domestic this respect product is still blank at present.
Three, summary of the invention
The object of the invention provides a kind of preparation and uses the method that the NJU-Fhhh1 specific bacteria is efficiently handled waste water.Especially the genetically engineered special efficacy bacterium NJU-Fhhh1 that uses the originality of the present invention's structure is a starting strain, preparation NJU-Fhhh1 specific bacteria, have the high degradability of white-rot fungi, the high-adaptability of native bacterium YZ1, the high flocculence of yeast saccharomyces cerevisiae, be beneficial to efficient processing organic waste water.The object of the invention also is this specific bacteria, and cost is low, can widely use for domestic manufacturer, and supporting relevant utilisation technology, persistence is arranged at home.
Technical spirit of the present invention: with original special efficacy bacterium, fermentative preparation becomes to handle the specific bacteria of waste water.
(1). technical conceive: the genetically engineered special efficacy bacterium NJU-Fhhh1 of the originality that makes up with this problem is for setting out
Bacterial strain, the NJU-Fhhh1 specific bacteria of waste water is handled in preparation, has high degradability, the soil of white-rot fungi
The high-adaptability of work bacterium YZ1, the high flocculence of yeast saccharomyces cerevisiae are beneficial to efficient processing organic waste water. exists
Domestic production NJU-Fhhh1 specific bacteria, cost only are about 1/4 of import, and domestic producer uses
Rise.Supporting relevant utilisation technology has persistence at home.
(2). technology constitutes:
Starting strain NJU-Fhhh1 special efficacy bacterium: the original scientific payoffs that is this seminar.NJU-Fhhh1 special efficacy bacterium is merged by the protoplastis of white-rot fungi (parental plant 1), native bacterium YZ1 (parental plant 2), three parental plant thalline of yeast saccharomyces cerevisiae (parental plant 3), at same intracellular recombination and integration, makes up the genetic engineering bacterium that obtains by gene.NJU-Fhhh1 special efficacy bacterium has been concentrated high degradability, the high-adaptability of three parental plants, three high advantages of high flocculence, is beneficial to efficient processing organic waste water.There is not the gene contamination problem in the new bacterial strain that internationally recognized protoplastis constructs, so there is not the gene contamination problem in the NJU-Fhhh1 specific bacteria yet.
NJU-Fhhh1 special efficacy bacterium is formed by parental plant 1 white-rot fungi (Phanerochaete chrysosporium), parental plant 2 native bacterium YZ1 (Pseudomonas), the fusion of parental plant 3 yeast saccharomyces cerevisiaes (Saccharomyces cerevisiae) protoplastis.Prepare earlier: slough the cell walls of fungi or slough cell walls, the generation protoplastis of bacterium and after centrifugal collection protoplastis, buffer solution for cleaning, obtain with N,O-Diacetylmuramidase with helicase through protoplastis, carrying out secondary then merges, merge for the first time: the protoplastis of the parental plant 1 white-rot fungi cell of equivalent and parental plant 2 native bacterium YZ1 cells mix and polyoxyethylene glycol (PEG, MW=6000) and CaCl
2, sucrose preparation induced liquid in merge, collect after centrifugal after fusion product, the high osmotic buffer eccentric cleaning separate application on SIM1, SIM2, three kinds of solid differential mediums of SIM3,30 ℃ cultivated 7 days, the bacterium colony that can grow on three kinds of substratum simultaneously is the product Fhh that the first time, parents were merged transboundary; And parental plant 1 white-rot fungi cell and parental plant 2 native bacterium YZ1 cells can only be grown on SIM1 and SIM2 respectively; Merge for the second time: the protoplastis of the Fhh of the equivalent that obtained last time and parental plant 3 brewing yeast cells mixes, polyoxyethylene glycol (PEG, MW=6000) and CaCl
2, the sucrose preparation induced liquid in merge, after collecting fusion product after centrifugal, containing sucrose high osmotic buffer eccentric cleaning, separate application on SIM1, SIM2, three kinds of solid differential mediums of SIM3, the bacterium colony that can grow on SIM1, SIM2, three kinds of substratum of SIM3 simultaneously is product NJU-Fhhh1 or the Fhh that three parents are transboundary merged.
SIM1=SM+100u Streptomycin sulphate/ml (streptomycin, Sm);
SIM2=SM+100u nystatin/ml (nystatin, Nt);
SIM3=SM+100u Streptomycin sulphate/ml+100u nystatin/ml (Sm+Nt).
The liquid nutrient medium of preparation NJU-Fhhh1 specific bacteria: among the 1000ml: K
2HPO
43g; KH
2PO
41g; NH
4NO
30.5g; Na
2SO
40.1g; MgSO
47H
2O 10mg; MnSO
44H
2O 1mg; CaCl
20.5mg; FeSO
47H
2O 1mg; CH
3COONa 5g; Yeast extract 5g; Peptone 10g; Glucose 10g; 200g potato diffusion juice is transferred pH to 7.0; 121 ℃, 103kPa, moist heat sterilization 20min.The suitableeest culture temperature: 35 ℃; Optimal pH: 7.0.
The zymotechnique of preparation NJU-Fhhh1 specific bacteria:
Fermentation equipment: full automatic control bio-reactor;
Zymotechnique: continous-stable fermentation system; Leavening temperature, 33 ± 2 ℃; The pH value, 7.0 ± 1; Reaction
Dissolved oxygen in the device, 〉=2mg/L; , mineral salt flow, 0.001-0.005V/ (V.d); Cell concentration,
2-10g/L; In containing C, P, N nutrient solution, cultivate.
Table 1.NJU-Fhhh1 specific bacteria fermentation parameter
Processing parameter numerical value processing parameter numerical value T, leavening temperature, ℃ 33 ± 2 Sp, nutrient solution P source concentration, gTP/L 0.10P, tank pressure, (1 normal atmosphere=10
55 * 10
5＜Sf, C/N/P nutrient solution stream 0.33Pa), Pa amount, V/ (V.d) Fa, clean air air demand, 75≤Mr, mineral salt flow, V/ (V.d) 0.003m
3/ kgCODRr, mixing speed, rpm 1000 X, cell concentration, g/L 4.99PH 7.0 ± 1 HRT, reaction solution fermentation time, h 8DO, dissolved oxygen in the reactor, mg/L 〉=2 Fr adds silicon oil foam killer flow velocity Sf＜Va, the reaction solution loading amount, % 80≤μ, than rate of increase, mg/mgL h
-10.1 27So, nutrient solution C source concentration, gCOD/L 8.48 q, nutrient solution are than utilization ratio, h
-10.211Sn, nutrient solution N source concentration, gTN/L 0.50 Yo, thalline output rate coefficient, % 59 beneficial effect of the invention:
(1) production cost of NJU-Fhhh1 specific bacteria is low, only is about 500 yuan/ton, and price can be far below the microbial inoculum of import;
(2) the NJU-Fhhh1 specific bacteria has three high advantages of three parental plants; Improve processing efficiency and reduce expenses more than 25%;
(3) the NJU-Fhhh1 specific bacteria both can be handled poisonous organic wastewater, also can handle conventional organic waste water;
(4) the NJU-Fhhh1 specific bacteria is used protoplastis fusion engineering bacteria, does not produce new gene, does not have the gene contamination problem.
In a word, microbial inoculum of the present invention improves degrading waste water efficient and saves working cost more than 25% than indigenous bacterium.The present invention uses the original special efficacy bacterium that protoplastis merges, and is the first that the waste water specific bacteria is handled in preparation.Especially use the special efficacy bacterium with international originality, the specific bacteria of preparation has the advantage of domestic popularization on cost and price.Organic waste waters such as degraded pure terephthalic acid (PTA) petrochemical wastewater have high degraded, high flocculation, high adaptation advantage.
Four, description of drawings
Fig. 1 is parental plant 1 white-rot fungi of the present invention (Phanerochaete chrysosporium) electron micrograph
Fig. 2 is parental plant 2 native bacterium YZ1 (Pseudomonas) of the present invention, electron micrograph
Fig. 3 is parental plant 3 yeast saccharomyces cerevisiaes of the present invention (Saccharomyces cerevisiae) electron micrograph
Fig. 4 is the NJU-Fhhh1 bacterial strain electron micrograph figure that obtains after the present invention's three strains are merged
Five, embodiment
The zymotechnique flow process of NJU-Fhhh1 specific bacteria technology of preparing
Example title: preparation of NJU-Fhhh1 specific bacteria and degraded pure terephthalic acid PTA petrochemical wastewater thereof
(1) application NJU-Fhhh1 specific strain prepares the condition control preparation microbial inoculum of NJU-Fhhh1 specific bacteria
Bio-reactor: Germany produces the full automatic control bio-reactor of B.Braun, useful volume 1L; Automatic control parameter setting: DO (dissolved oxygen)=2mg/L; PH=7.0; T=35 ℃; Sf (liquid nutrient medium flow)=0.33 (V/V);
Mixing speed: 1000rpm; Foam-expelling: silicone oil; Liquid level: reach the 1L useful volume; Mineral salt flow: 0.003 (V/V); Liquid nutrient medium: be same as the aforesaid liquid substratum
(2) the parametric measurement NJU-Fhhh1 special efficacy thalline biomass concentration X:4.99g/L of NJU-Fhhh1 specific bacteria prepared product; Than rate of increase μ: 0.127h
-1Substratum than utilization ratio q:0.211h
-1Yield coefficient Yo:59.84%; Substrate residual concentration Se:0.15g (COD)/L. in the preparation liquid
(3) the preservation prescription of NJU-Fhhh1 specific bacteria prepared product (4 ℃)
Prescription 1:NJU-Fhhh1 specific bacteria fermented liquid does not add any other composition;
Prescription 2:NJU-Fhhh1 specific bacteria fermented liquid+antioxidant mercaptoethanol (SH); (final concentration 0.2 ‰);
Prescription 3:NJU-Fhhh1 specific bacteria fermented liquid+Fe
3+, (final concentration 0.14%);
Prescription 4:NJU-Fhhh1 specific bacteria fermented liquid+Fe
3++ mercaptoethanol (SH); (final concentration 0.2 ‰);
(4) best NJU-Fhhh1 specific bacteria degraded pure terephthalic acid PTA petrochemical wastewater parameter of preserving prescription: the preservation of process 2002/5/11-2002/7/21 70 days (4 ℃), product+antioxidant mercaptoethanol (SH) is an optimum formula to measure prescription 2:NJU-Fhhh1 specific bacteria original fermented solution body.Degraded pure terephthalic acid PTA petrochemical wastewater parameter is: NJU-Fhhh1 special efficacy thalline high specific rate of increase μ
Max, be 0.067
h -1High specific degradation rate q
Max, be 0.226
h -1Yield coefficient Y is 29.64%; Self reduction coefficient is 0.0026
h -1
(5) the NJU-Fhhh1 specific bacteria prepares case summary
The zymotechnique control condition of preparation specific bacteria is: DO (dissolved oxygen)=2mg/L; PH=7.0; T=35 ℃; Sf (liquid nutrient medium flow)=0.33 (V/V); Mixing speed: 1000rpm; The silicone oil foam-expelling; Mineral salt flow: 0.003 (V/V); The top condition of preserving the NJU-Fhhh1 specific bacteria is: 4 ℃ of preservations, special efficacy fermented liquid+mercaptoethanol (SH);
The NJU-Fhhh1 specific bacteria is separated pure terephthalic acid's petrochemical wastewater parameter: μ
Max=0.067
h -1High specific degradation rate q
Max=0.226
h -1Yield coefficient Y is 29.64%; Self reduction coefficient is 0.0026
h -1The NJU-Fhhh1 specific bacteria has high degraded, high flocculation, high adaptation advantage; Raise the efficiency and reduce expenses more than 25%.
The Fhhh1 specific bacteria is handled the applied research result of waste water example
Kind of waste water PTA waste water stripping suede waste water frigon waste water
2001 Jinhu County, 2002 Zhangjiagangs, 2002 Nanjing, time place
Technology activated sludge process activated sludge process activated sludge process raw wastewater COD, kg/m
34.344 1.030 9.45 raw wastewater BOD, kg/m
32.828 0.4777 8.18 raw wastewater SS, kg/m
30.103 0.035 21.69 raw wastewater pH 4.9＞14 12 raw wastewater flows, m
3/ d 10,000 960 8 waste water reaction temperature, ℃ 35 35 35Yobs, % 0.1976 0.4638 0.6687Qmax, d
-10.226 1.078 22.46Ksq, kg/m
30.01 0.1 2.70 μ max, d
-10.067 0.6 15.02Ks μ, kg/m
30.02 0.02 2.7Yt, % 0.2046 0.5566 0.6688Kd, d
-10.002 0.0455 0.0001q, d
-10.113 0.4000 0.1651 μ, d
-10.0223 0.8624 0.1104 water outlet COD, mg/L 0.01 0.04 0.02 water outlet SS, mg/L 0.0005 0.0818 0.067 water outlet pH 7.0 7.0 7.0Qr, m
3/ d 10,000 90 8Xr, m
3/ d 0.57 0.76 5.35 Θ c, d 44.78 2.5 9.05HRT, d 0.04 1.34 0.11X, kg/m
30.2855 the volume required V of 0.1399 2.708 indigenous bacterium, m
3The volume required V of 2,293 1900 156Fhhh, m
31,624 1524 19.4Fhhh save construction investment, and % 29.18 19.79 87.56
Microbial preservation data of the present invention: on July 26th, 2002, the guarantor subtracted, and deposit number is that to merge the special efficacy bacterium transboundary be white-rot fungi (phanerochaete chrysoporium) yeast saccharomyces cerevisiae (saccharomyces cerevisiae), native bacterium (YZ1) three protoplastis fusion product to CGMCCNo.0783 three parental plants.China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, address: No. 13 Institute of Microorganism, Academia Sinica in north, ZhongGuancun, Haidian District, BeiJing.
Claims (8)
1, a kind of preparation method of specific bacteria, it is characterized in that merging by the protoplastis of white-rot fungi (parental plant 1), native bacterium YZ1 (parental plant 2), three parental plant thalline of yeast saccharomyces cerevisiae (parental plant 3), at same intracellular recombination and integration, make up the genetic engineering bacterium that obtains by gene.Its fermentation parameter is: leavening temperature, 33 ± 2 ℃; The pH value, 7.0 ± 1; Dissolved oxygen in the reactor, 〉=2mg/L; The mineral salt flow, 0.001-0.005V/ (V.d); Cell concentration, 2-10g/L; In containing C, P, N nutrient solution, cultivate.
2,, it is characterized in that described mineral salt comprises Na by the preparation method of the described specific bacteria of claim 1
2SO
4, MgSO
47H
2O, MnSO
44H
2O, CaCl
2, FeSO
47H
2O.
3, by the preparation method of the described specific bacteria of claim 1, the composition that it is characterized in that preparing the liquid nutrient medium of NJU-Fhhh1 specific bacteria is: among the 1000ml: K
2HPO
43g; KH
2PO
41g; NH
4NO
30.5g; Na
2SO
40.1g; MgSO
47H
2O 10mg; MnSO
44H
2O 1mg; CaCl
20.5mg; FeSO
47H
2O 1mg; CH
3COONa 5g; Yeast extract 5g; Peptone 10g; Glucose 10g; 200g potato diffusion juice is transferred pH to 7.0; 121 ℃, 103kPa, moist heat sterilization 20min.
4, by the preparation method of the described specific bacteria of claim 1, it is characterized in that the suitableeest culture temperature: 35 ℃; Optimal pH: 7.0.
5,, it is characterized in that the preservation prescription of NJU-Fhhh1 specific bacteria prepared product, at antioxidant mercaptoethanol (SH) by the preparation method of the described specific bacteria of claim 1; (final concentration 0.1-0.3 ‰);
6,, it is characterized in that the preservation prescription of NJU-Fhhh1 specific bacteria prepared product, original fermented solution body product+Fe by the preparation method of the described specific bacteria of claim 1
3+, (final concentration 0.14%).
7,, it is characterized in that the preservation prescription of NJU-Fhhh1 specific bacteria prepared product, original fermented solution body product+Fe by the preparation method of the described specific bacteria of claim 1
3++ antioxidant mercaptoethanol (SH).
8, by the described specific bacteria of claim 1 handle wastewater from chemical industry or conventional organic waste water method, it is characterized in that being used to degrade pure terephthalic acid's petrochemical wastewater, stripping suede waste water, frigon waste water.
Priority Applications (1)
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|---|---|---|---|
| CNB021381712A CN100497618C (en) | 2002-08-26 | 2002-08-26 | Specific bacteria preparation and method for treating chemical waste water or conventional organic waste water |
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| CNB021381712A CN100497618C (en) | 2002-08-26 | 2002-08-26 | Specific bacteria preparation and method for treating chemical waste water or conventional organic waste water |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100335426C (en) * | 2004-10-22 | 2007-09-05 | 扬子石油化工股份有限公司 | Technological process for treating PTA petrochemical waste water by using specific strain |
| CN100355875C (en) * | 2004-12-17 | 2007-12-19 | 中国科学院成都生物研究所 | Microbe bacteria agent for treatment of refinery waste water, its preparation method and application |
| CN106277579A (en) * | 2016-08-15 | 2017-01-04 | 吴小慧 | The deep treatment method of cultivation factory sewage |
| CN107117663A (en) * | 2017-03-31 | 2017-09-01 | 长乐巧通工业设计有限公司 | A kind of environment-protecting industrial waste water treating agent and preparation method thereof |
-
2002
- 2002-08-26 CN CNB021381712A patent/CN100497618C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100335426C (en) * | 2004-10-22 | 2007-09-05 | 扬子石油化工股份有限公司 | Technological process for treating PTA petrochemical waste water by using specific strain |
| CN100355875C (en) * | 2004-12-17 | 2007-12-19 | 中国科学院成都生物研究所 | Microbe bacteria agent for treatment of refinery waste water, its preparation method and application |
| CN106277579A (en) * | 2016-08-15 | 2017-01-04 | 吴小慧 | The deep treatment method of cultivation factory sewage |
| CN107117663A (en) * | 2017-03-31 | 2017-09-01 | 长乐巧通工业设计有限公司 | A kind of environment-protecting industrial waste water treating agent and preparation method thereof |
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| Publication number | Publication date |
|---|---|
| CN100497618C (en) | 2009-06-10 |
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