CN101130754A - High-density ferment method for petroleum hydrocarbon degradation bacterium - Google Patents
High-density ferment method for petroleum hydrocarbon degradation bacterium Download PDFInfo
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- CN101130754A CN101130754A CNA200710119858XA CN200710119858A CN101130754A CN 101130754 A CN101130754 A CN 101130754A CN A200710119858X A CNA200710119858X A CN A200710119858XA CN 200710119858 A CN200710119858 A CN 200710119858A CN 101130754 A CN101130754 A CN 101130754A
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Abstract
The invention discloses a high-density fermenting method of petroleum hydrocarbon degraded bacteria in the biological fermenting technical domain, which is characterized by the following: activating slope bacteria; enlarging the cultured seed liquid; preparing before inoculating; inoculating; supplementing the ferment culture medium batch by batch; adjusting the even growing speed of bacteria; reducing cost; simplifying the operation; producing large amount of petroleum hydrocarbon degraded bacteria within short period; solving the problem of soil pollution and the accident of petroleum pollution.
Description
Technical field
The invention belongs to technical field of biological fermentation, particularly a kind of fermentation process in high density of petroleum hydrocarbon degradation bacterium.
Background technology
Bioremediation technology is the main recovery technique of oil and related products contaminated soil.But form complicated, bio-refractory and have lower characteristics such as biology availability because petroleum pollution has, indigenous microorganism is difficult to effectively, the petroleum hydrocarbon in the soil of degrading fast, completely.Biological additive process can have the efficient of the microorganism raising biological restoration of efficient degradation petroleum hydrocarbon ability by interpolation, effectively removes petroleum hydrocarbon contaminant.Yet improvement for the unexpected accident of the improvement of extensive oil-polluted soils or petroleum pollution, can bring the heavy demand of the microorganism of efficient degradation petroleum hydrocarbon ability, therefore the process exploitation that adopts high density fermentation to produce petroleum hydrocarbon degradation bacterium seems particularly urgent.The scientific research personnel of the biological institute in Chinese Academy of Sciences Chengdu in 2006 has born country " 863 " plan water pollution control technology and two problems of administering in the great special project of engineering---optimization regulation and control and efficient anaerobic and the aerobe reactor development and application (reagent and fine chemicals .2006 (2) .-3-3) of microbial population in efficient excellent species seed selection and the treatment system.Only mention that just oil and dyeing waste water are main a plurality of strain microbial inoculums and high density fermentation thereof, do not have concrete zymotechnique route, do not have concrete output.Do not find relevant report abroad.For small-scale engineering construction, the bacterium microbial inoculum that applies in the process of the test generally is to shake bottle directly to cultivate in the laboratory, then microbial inoculum is transported to the testing ground and applies at present.On the one hand, can't satisfy the requirement of producing a large amount of microbial inoculum amounts at short notice that more large-scale industry is implemented; On the other hand, increase the cost of biological treating, reduced the efficient of biological treating.
Summary of the invention
The object of the present invention is to provide a kind of fermentation process in high density of petroleum hydrocarbon degradation bacterium, it is characterized in that, this method is to utilize high density fermentation to produce the method for petroleum hydrocarbon degradation bacterium, prepare seed liquor, the preceding preparation of inoculation and inoculation, fed-batch fermentation cultivation through slant strains activation, enlarged culturing, the average growth velocity of regulation and control thalline is produced petroleum hydrocarbon degradation bacterium with high density fermentation.This method cost is low, simple to operate, can produce bulk petroleum alkane degradation bacterium in the short period of time, has solved the bacterial classification problem of the improvement of extensive oil-polluted soils improvement and the unexpected accident of petroleum pollution.
This preparation method comprises following processing step:
(1) slant strains is activated, prepares fermentation seed liquid through dull and stereotyped LB culture medium culturing;
(2) seed liquor of preparation in the step (1) is inoculated in fermention medium in the 5-6% ratio of fermention medium volume and ferments, prepare and inoculation before comprising fermentation.Wherein batch fermentation substratum and fed-batch fermentation substratum comprise the component shown in the table 1 (g/L):
Table 1 batch fermentation substratum and fed-batch fermentation nutrient media components
| Composition | The batch fermentation substratum, g/L | Supplemented medium, g/L |
| Glucose yeast powder K 2HPO 4 NaH 2PO 4 (NH 4) 2SO 4 MgSO 4·7H 2O EDTA trace element | 30.0 50.0 8.7 4.2 5.5 2.5 1 1ml | 1000.0 400.0 26.0 12.6 16.5 20 3 3ml |
Culture temperature is 32～37 ℃, and the average growth velocity of thalline is controlled at 0.2-0.4h
-1
The pH value is controlled at 6.8 in the fermenting process, and carries out feed supplement, and the wear rate of control feed rate and thalline is suitable, and fermentation finishes, and collects fermented liquid, and glycerol adding is preserved.Fermentation period is 12-14 hour;
(3) adopt in batches-the feed supplement training method, pH, dissolved oxygen control before the feed supplement;
(4) the feed supplement stage
1. stream adds glucose solution: during the fermentation, when the C source exhausted, promptly the horizontal DO of dissolved oxygen rose in rain, and pH rises in rain, stream adds glucose solution, and giving and mending concentration in the fermentor tank is that the glucose solution of 1000g/L makes that glucose concn is controlled in the 0.2-0.5g/L scope in the fermentor tank;
2. stream adds the yeast soln that contains fermentation inorganic salt and trace element: when the N source exhausted, promptly the horizontal D0 of dissolved oxygen rose in rain, and pH descends in rain, and stream adds the yeast soln that contains fermentation inorganic salt and trace element;
The preparation process of described fermentation seed liquid is: choose the bacterium colony of 2-3 after activated from plate culture medium, place the 300mL that fills 50mL first order seed substratum to shake bottle, in temperature is 32～37 ℃, and rotating speed is to cultivate 8-10h in the shaking table of 200rpm, obtains nutrient solution; Again above-mentioned seed liquor is divided equally four 300mL that fill the 50mL secondary seed medium and shake in the bottle, under identical condition, cultivate 10-12h, promptly get fermentation seed liquid.
Prepare before the described fermentation and inoculation: the batch fermentation substratum except that glucose is dissolved in the 3.5L deionized water, pours in the fermentor tank, proofread and correct the pH instrument, sterilization, proofreading and correct dissolved oxygen D0 in the sterilization process is 0%; Under the protection of flame circle; with glucose solution and with the inoculum size of the 5-6% of fermention medium with petroleum hydrocarbon degradation bacterium (enterobacter cloacae; Enterobacte cloacae; this laboratory screens from the contaminated soil of Zhongyuan Oil Field, and preserve) secondary seed is inoculated in the above-mentioned fermentor tank; 32～37 ℃ of culture temperature; rotating speed 300rpm proofreaies and correct dissolved oxygen and reaches 100%.
PH, dissolved oxygen control are being controlled pH6.5 ± 0.1 in early stage with ammoniacal liquor before the described feed supplement, and it is 6.5 too many that pH can not be higher than, otherwise magnesium ion can precipitate, thereby are unfavorable for thalli growth; Also avoid thalli growth to produce acidic substance and made pH descend, influenced thalli growth.When entering feed supplement, pH is set in 6.8.Dissolved oxygen is controlled at more than 20%, improves dissolved oxygen level by the mode that improves rotating speed, mentions 1200rpm until rotating speed.
Processing condition and processing parameter concrete in each processing step of the present invention are:
The trace element of described step (2) is formed (g/L): H by following composition
3BO
32.85, MnSO
4H
2O1.55, FeSO
47H
2O 1.362, Seignette salt 1.77, CuCl
22H
2O 0.0265, ZnSO
47H
2O0.044, CoCl
46H
2O 0.0405, Na
2MoO
42H
2O 0.0251.
Dull and stereotyped bacterial classification in the described step (1) is prepared into seed liquor through the secondary enlarged culturing, and wherein fermention medium consists of (g/L) in the first step enlarged culturing: glucose 10, and Tryptones 10, yeast extract 5, NaCl 10.Culture temperature is 34 ℃, and rotating speed is to cultivate 8-10h in the shaking table of 200rpm.
Fermention medium consists of (g/L) in the enlarged culturing of the described second stage: glucose 10, yeast extract 5, KH
2PO
41, K
2HPO
41, NH
4NO
41, MgSO
47H
2O 0.5.Culture temperature is 34 ℃, and rotating speed is to cultivate 10-12h in the shaking table of 200rpm.
Training method in the described step (2) is the batch feeding training method, and the feed supplement mode adopts nearly index fed-batch mode, and wherein batch fermentation substratum and fed-batch fermentation substratum comprise the component shown in the table 2 (g/L)
Table 2 batch fermentation substratum and fed-batch fermentation nutrient media components
| Composition | The batch fermentation substratum, g/L | Supplemented medium, g/L |
| Glucose | 30.0 | 1000.0 |
| Yeast powder K 2HPO 4 NaH 2PO 4 (NH 4) 2SO 4 MgSO 4·7H 2O EDTA trace element | 50.0 8.7 4.2 5.5 2.5 1 1ml | 400.0 26.0 12.6 16.5 20 3 3ml |
The invention has the beneficial effects as follows and utilize high density fermentation to produce petroleum hydrocarbon degradation bacterium.Thereby provide a large amount of degradation bacteria to oil-polluted soils original position or heterotopic biological reparation, improved degradation efficiency, reduced the biological restoration cost.Adopt petroleum hydrocarbon degradation bacterium high density fermentation method of the present invention, the biomass of petroleum hydrocarbon degradation bacterium is improved largely.By high density fermentation, make cell accumulation volume OD
600Value reaches about 60.Reduce the production cycle, lowered production cost.Fermentation technology process of the present invention is simple, and easy handling has broad application prospects in the biological restoration field of oil-polluted soils.
Description of drawings
Fig. 1 is a mixing speed and dissolved oxygen change curve in time.
Fig. 2 is base consumption and cell growth curve figure.
Embodiment
The invention provides a kind of fermentation process in high density of petroleum hydrocarbon degradation bacterium.This method is to utilize high density fermentation to produce the method for petroleum hydrocarbon degradation bacterium, following processing step:
(1) slant strains is activated, prepares fermentation seed liquid through dull and stereotyped LB culture medium culturing;
(2) seed liquor of preparation in the step (1) is inoculated in fermention medium in the 5-6% ratio of fermention medium and ferments, prepare and inoculation before comprising fermentation.Wherein batch fermentation substratum and fed-batch fermentation substratum comprise the component shown in the table 1 (g/L):
Table 1 batch fermentation substratum and fed-batch fermentation nutrient media components
| Composition | The batch fermentation substratum, g/L | Supplemented medium, g/L |
| Glucose yeast powder K 2HPO 4 NaH 2PO 4 | 30.0 50.0 8.7 4.2 | 1000.0 400.0 26.0 12.6 |
| (NH 4) 2SO 4 MgSO 4·7H 2O EDTA trace element | 5.5 2.5 1 1ml | 16.5 20 3 3ml |
Culture temperature is 32～37 ℃, and the average growth velocity of thalline is controlled at 0.2-0.4h
-1
The pH value is controlled at 6.8 in the fermenting process, and carries out feed supplement, and the wear rate of control feed rate and thalline is suitable, and fermentation finishes, and collects fermented liquid, and glycerol adding is preserved.Fermentation period is 12-14 hour;
(3) adopt in batches-the feed supplement training method, pH, dissolved oxygen control before the feed supplement;
(4) the feed supplement stage
1. stream adds glucose solution: during the fermentation, when the C source exhausted, promptly the horizontal DO of dissolved oxygen rose in rain, and pH rises in rain, stream adds glucose solution, and giving and mending concentration in the fermentor tank is that the glucose solution of 1000g/L makes that glucose concn is controlled in the 0.2-0.5g/L scope in the fermentor tank;
2. stream adds the yeast soln that contains fermentation inorganic salt and trace element: when the N source exhausted, promptly the horizontal DO of dissolved oxygen rose in rain, and pH descends in rain, and stream adds the yeast soln that contains fermentation inorganic salt and trace element;
The preparation process of described fermentation seed liquid is: choose the bacterium colony of 2-3 after activated from plate culture medium, place the 300mL that fills 50mL first order seed substratum to shake bottle, in temperature is 32～37 ℃, and rotating speed is to cultivate 8-10h in the shaking table of 200rpm, obtains nutrient solution; Again above-mentioned seed liquor is divided equally four 300mL that fill the 50mL secondary seed medium and shake in the bottle, under identical condition, cultivate 10-12h.Promptly get fermentation seed liquid.
Prepare before the described fermentation and inoculation: the batch fermentation substratum except that glucose is dissolved in the 3.5L deionized water, pours in the fermentor tank, proofread and correct the pH instrument, sterilization, proofreading and correct dissolved oxygen DO in the sterilization process is 0%; Under the protection of flame circle; with glucose solution and with the inoculum size of the 5-6% of fermention medium with petroleum hydrocarbon degradation bacterium (enterobacter cloacae; Enterobacte cloacae; this laboratory screens from the contaminated soil of Zhongyuan Oil Field, and preserve) secondary seed is inoculated in the above-mentioned fermentor tank; 32～37 ℃ of culture temperature; rotating speed 300rpm proofreaies and correct dissolved oxygen and reaches 100%.
PH, dissolved oxygen control are being controlled pH6.5 ± 0.1 in early stage with ammoniacal liquor before the described feed supplement, and it is 6.5 too many that pH can not be higher than, otherwise magnesium ion can precipitate, thereby are unfavorable for thalli growth; Also avoid thalli growth to produce acidic substance and made pH descend, influenced thalli growth.When entering feed supplement, pH is set in 6.8.Dissolved oxygen is controlled at more than 20%, improves dissolved oxygen level by the mode that improves rotating speed, mentions 1200rpm until rotating speed.
Processing condition and processing parameter concrete in each processing step of the present invention are:
The trace element of described step (2) is formed (g/L): H by following composition
3BO
32.85, MnSO
4H
2O1.55, FeSO
47H
2O 1.362, Seignette salt 1.77, CuCl
22H
2O 0.0265, ZnSO
47H
2O0.044, CoCl
46H
2O 0.0405, Na
2MoO
42H
2O 0.0251.
Dull and stereotyped bacterial classification in the described step (1) is prepared into seed liquor through the secondary enlarged culturing, and wherein fermention medium consists of (g/L) in the first step enlarged culturing: glucose 10, and Tryptones 10, yeast extract 5, NaCl 10.Culture temperature is 34 ℃, and rotating speed is to cultivate 8-10h in the shaking table of 200rpm.
Fermention medium consists of (g/L) in the enlarged culturing of the described second stage: glucose 10, yeast extract 5, KH
2PO
41, K
2HPO
41, NH
4NO
41, MgSO
47H
2O 0.5.Culture temperature is 34 ℃, and rotating speed is to cultivate 10-12h in the shaking table of 200rpm.
Training method in the described step (2) is the batch feeding training method, and the feed supplement mode adopts nearly index fed-batch mode, and wherein batch fermentation substratum and fed-batch fermentation substratum comprise the component shown in the table 2 (g/L)
Table 2 batch fermentation substratum and fed-batch fermentation nutrient media components
| Composition | The batch fermentation substratum, g/L | Supplemented medium, g/L |
| Glucose yeast powder K 2HPO 4 NaH 2PO 4 (NH 4) 2SO 4 MgSO 4·7H 2O EDTA trace element | 30.0 50.0 8.7 4.2 5.5 2.5 1 1ml | 1000.0 400.0 26.0 12.6 16.5 20 3 3ml |
Enumerating embodiment is below further specified.
Example one: a kind of fermentation process in high density of petroleum hydrocarbon degradation bacterium
This petroleum hydrocarbon efficient degrading bacteria is that this laboratory is screened from the contaminated soil of Zhongyuan Oil Field and preserved.
1. seed culture
Choose the bottle that shakes that the bacterium colony of 2-3 after activated places a 300mL who fills 50mL first order seed substratum from plate culture medium, temperature is 34 ℃, and rotating speed is to cultivate 8-10h in the shaking table of 200rpm, obtains the first order seed nutrient solution; Again the first order seed nutrient solution is divided equally the shaking in the bottle of four 300mL that fill the 50mL secondary seed medium, under identical condition, cultivated 10-12h.Promptly get fermentation seed liquid.
2. prepare before planting
Batch fermentation substratum except that glucose is dissolved in the 3.5L deionized water, pours in the clean fermentor tank, proofread and correct the pH instrument, sterilization, proofreading and correct dissolved oxygen DO in the sterilization process is 0%, connects air filter and other auxiliary facilitys.
3. inoculation
Under flame circle protection, with glucose solution with the inoculum size of 5-6% fermentation seed liquid is inoculated in 5L and controls automatically in the fermentor tank, liquid amount 3.5L, ventilation 5L/min, stirring velocity 300rpm, 34 ℃ of culture temperature are proofreaied and correct dissolved oxygen and are reached 100%.Control pH6.5 ± 0.1 with ammoniacal liquor early stage, and it is 6.5 too many that pH can not be higher than, otherwise magnesium ion can precipitate, thereby be unfavorable for thalli growth; Also avoid thalli growth to produce acidic substance and made pH descend, influenced thalli growth.Dissolved oxygen is controlled at more than 20%, improves dissolved oxygen level by the mode that improves rotating speed, mentions 1200rpm until rotating speed.
4. feed supplement growth phase
When rotating speed transferred to maximum fast 1200rpm, the DO value descended, and keeps.When being exhausted of nutritive substance, DO can rise rapidly, because the C source runs out of earlier, so rising along with DO, the pH value can rise rapidly, need carry out feed supplement this moment, and giving and mending concentration in the fermentor tank is that the glucose solution of 1000g/L makes that glucose concn is controlled in the 0.2-0.5g/L scope in the fermentor tank.The automatic controlling flow of pH adds ammoniacal liquor and regulates pH 6.8.Along with being exhausted of N source, DO can rise rapidly, and this moment, the pH value can descend rapidly, and explanation will add the N source by stream.
Fig. 1 has shown the process relation of control dissolved oxygen and rotating speed.Make the DO value remain on more than 20% early stage as far as possible.
(1) stream adds glucose solution: during the fermentation, when exhausting, the C source (inoculates back 6 hours approximately), be that the horizontal DO of dissolved oxygen rises in rain, pH rises in rain, stream adds glucose solution, and giving and mending concentration in the fermentor tank is that the glucose solution of 1000g/L makes that glucose concn is controlled in the 0.2-0.5g/L scope in the fermentor tank;
(2) stream adds the yeast soln that contains fermentation inorganic salt and trace element: after approximately inoculating back 6.8 hours, when the N source exhausted, promptly the horizontal DO of dissolved oxygen rose in rain, and pH descends in rain, and stream adds the yeast soln that contains fermentation inorganic salt and trace element.
Fig. 2 has shown the relation of base consumption and cell growth.Preceding 6 hours of fermentation culture, bacterial concentration rises rapidly, flex point appears in growth curve when 6hr, illustrate that the fermenting bacteria growth is subjected to the intact influence of base consumption, the beginning batch feeding makes a jar interior glucose concn be controlled at the 0.2-0.5g/L bacterium and enters the metastable rise period, up to fermentation ends, dissolved oxygen is near exhausting, and this moment, bacterial concentration reached maximum value.By high density fermentation, make cell accumulation volume OD
600Value reaches about 60.
The present invention is used for the biological restoration field of oil-polluted soils, has broad application prospects.By the production technique of high density fermentation of exploitation petroleum hydrocarbon degradation bacterium, reduce rehabilitation cost, improved repairing efficiency, and the recovery project that can accelerate extensive oil-polluted soils implement to provide safeguard.
Claims (8)
1. the fermentation process in high density of a petroleum hydrocarbon degradation bacterium, it is characterized in that, this method is to utilize high density fermentation to produce the method for petroleum hydrocarbon degradation bacterium, prepare seed liquor, the preceding preparation of inoculation and inoculation, fed-batch fermentation cultivation through slant strains activation, enlarged culturing, the average growth velocity of regulation and control thalline is produced petroleum hydrocarbon degradation bacterium with high density fermentation.
2. according to the fermentation process in high density of the described petroleum hydrocarbon degradation bacterium of claim 1, it is characterized in that this preparation method comprises following processing step:
(1) slant strains is activated, prepares fermentation seed liquid through dull and stereotyped LB culture medium culturing;
(2) seed liquor of preparation in the step (1) is inoculated in fermention medium in the 5-6% ratio of fermention medium volume and ferments, prepare and inoculation before comprising fermentation, wherein batch fermentation substratum and fed-batch fermentation substratum comprise the component shown in the table 1 (g/L):
Table 1 batch fermentation substratum and fed-batch fermentation nutrient media components
Culture temperature is 32～37 ℃, and the average growth velocity of thalline is controlled at 0.2-0.4h
-1
The pH value is controlled at 6.8 in the fermenting process, and carries out feed supplement, and the wear rate of control feed rate and thalline is suitable, and fermentation finishes, and collects fermented liquid, and glycerol adding is preserved; Fermentation period is 12-14 hour;
(3) adopt in batches-the feed supplement training method, pH, dissolved oxygen control before the feed supplement;
(4) the feed supplement stage:
1. stream adds glucose solution: during the fermentation, when the C source exhausted, promptly the horizontal DO of dissolved oxygen rose in rain, and pH rises in rain, stream adds glucose solution, and giving and mending concentration in the fermentor tank is that the glucose solution of 1000g/L makes that glucose concn is controlled in the 0.2-0.5g/L scope in the fermentor tank;
2. stream adds the yeast soln that contains fermentation inorganic salt and trace element: when the N source exhausted, promptly the horizontal DO of dissolved oxygen rose in rain, and pH descends in rain, and stream adds the yeast soln that contains fermentation inorganic salt and trace element.
3. according to the fermentation process in high density of the described petroleum hydrocarbon degradation bacterium of claim 1, it is characterized in that, the preparation process of described fermentation seed liquid is: choose the bacterium colony of 2-3 after activated from plate culture medium, place the 300mL that fills 50mL first order seed substratum to shake bottle, in temperature is 32～37 ℃, rotating speed is to cultivate 8-10h in the shaking table of 200rpm, obtains nutrient solution; Again above-mentioned seed liquor is divided equally four 300mL that fill the 50mL secondary seed medium and shake in the bottle, under identical condition, cultivate 10-12h.Promptly get fermentation seed liquid.
4. according to the fermentation process in high density of the described petroleum hydrocarbon degradation bacterium of claim 1, it is characterized in that, preparation and inoculation before the described fermentation: the batch fermentation substratum except that glucose is dissolved in the 3.5L deionized water, pour in the fermentor tank, proofread and correct the pH instrument, sterilization, proofreading and correct dissolved oxygen DO in the sterilization process is 0%; Under the protection of flame circle; with glucose solution and with the inoculum size of the 5-6% of fermention medium with petroleum hydrocarbon degradation bacterium (enterobacter cloacae; Enterobacte cloacae; this laboratory screens from the contaminated soil of Zhongyuan Oil Field, and preserve) secondary seed is inoculated in the above-mentioned fermentor tank; 32～37 ℃ of culture temperature; rotating speed 300rpm proofreaies and correct dissolved oxygen and reaches 100%.
5. according to the fermentation process in high density of the described petroleum hydrocarbon degradation bacterium of claim 2, it is characterized in that pH, dissolved oxygen control before the described feed supplement, control pH6.5 ± 0.1 in early stage with ammoniacal liquor, it is 6.5 too many that pH can not be higher than, otherwise magnesium ion can precipitate, thereby be unfavorable for thalli growth; Also avoided thalli growth to produce acidic substance and made pH descend, influenced thalli growth, when entering feed supplement, pH is set in 6.8, and dissolved oxygen is controlled at more than 20%, improves dissolved oxygen level by the mode that improves rotating speed, mentions 1200rpm until rotating speed.
6. according to the fermentation process in high density of the described petroleum hydrocarbon degradation bacterium of claim 2, it is characterized in that the trace element of described step (2) is formed (g/L): H by following composition
3BO
32.85, MnSO
4H
2O 1.55, FeSO
47H
2O 1.362, Seignette salt 1.77, CuCl
22H
2O 0.0265, ZnSO
47H
2O 0.044, CoCl
46H
2O 0.0405, Na
2MoO
42H
2O 0.0251.
7. according to the fermentation process in high density of the described petroleum hydrocarbon degradation bacterium of claim 2, it is characterized in that, dull and stereotyped bacterial classification in the described step (1) is prepared into seed liquor through the secondary enlarged culturing, wherein fermention medium consists of (g/L) in the first step enlarged culturing: glucose 10, Tryptones 10, yeast extract 5, NaCl 10; Culture temperature is 34 ℃, and rotating speed is to cultivate 8-10h in the shaking table of 200rpm;
Fermention medium consists of (g/L) in the enlarged culturing of the described second stage: glucose 10, yeast extract 5, KH
2PO
41, K
2HPO
41, NH
4NO
41, MgSO
47H
2O 0.5; Culture temperature is 34 ℃, and rotating speed is to cultivate 10-12h in the shaking table of 200rpm.
8. according to the fermentation process in high density of the described petroleum hydrocarbon degradation bacterium of claim 2, it is characterized in that, training method in the described step (2) is the batch feeding training method, the feed supplement mode adopts nearly index fed-batch mode, and wherein batch fermentation substratum and fed-batch fermentation substratum comprise the component shown in the table 2 (g/L).
Table 2 batch fermentation substratum and fed-batch fermentation nutrient media components
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| CN104726370A (en) * | 2015-02-16 | 2015-06-24 | 湖南农业大学 | Enterobacter with crude oil degradation effect and application of enteric bacilli |
| CN105312317A (en) * | 2015-05-08 | 2016-02-10 | 北京师范大学 | Method for repairing acid soil polluted by petroleum hydrocarbon and heavy metal with reinforced saccharomycetes |
| CN107299066A (en) * | 2017-06-21 | 2017-10-27 | 中国石油化工股份有限公司 | The preparation method and degradation process method of a kind of microbial degradation liquid of oily sludge |
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| CN110628672A (en) * | 2019-09-26 | 2019-12-31 | 中海石油环保服务（天津）有限公司 | Culture medium and high-density fermentation method of petroleum hydrocarbon degrading bacteria |
| CN111154702A (en) * | 2020-03-13 | 2020-05-15 | 辽宁大学 | Enterobacter cloacae for degrading petroleum pollution and application thereof |
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| CN1357617A (en) * | 2000-12-13 | 2002-07-10 | 中国科学院沈阳应用生态研究所 | Condensed oil polluted soil degrading bacteria and the usage |
| CN100355878C (en) * | 2004-12-17 | 2007-12-19 | 中国科学院成都生物研究所 | Alkane-degrading functional bacteria, its cultivation method and application |
| CN100371095C (en) * | 2005-12-21 | 2008-02-27 | 中国石油化工股份有限公司 | Heterotopic biological restoring method of petroleum hydrocarbon polluted soil |
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| CN104726370A (en) * | 2015-02-16 | 2015-06-24 | 湖南农业大学 | Enterobacter with crude oil degradation effect and application of enteric bacilli |
| CN105312317A (en) * | 2015-05-08 | 2016-02-10 | 北京师范大学 | Method for repairing acid soil polluted by petroleum hydrocarbon and heavy metal with reinforced saccharomycetes |
| CN107312515A (en) * | 2017-06-01 | 2017-11-03 | 大庆华理生物技术有限公司 | A kind of multi-element biologic compound oil displacement agent system and its injection technology |
| CN107312515B (en) * | 2017-06-01 | 2019-09-27 | 大庆华理生物技术有限公司 | A kind of multi-element biologic compound oil displacement agent system and its injection technology |
| CN107299066A (en) * | 2017-06-21 | 2017-10-27 | 中国石油化工股份有限公司 | The preparation method and degradation process method of a kind of microbial degradation liquid of oily sludge |
| CN107299066B (en) * | 2017-06-21 | 2020-08-28 | 中国石油化工股份有限公司 | Preparation method and degradation treatment method of microbial degradation liquid containing oily sludge |
| CN110628672A (en) * | 2019-09-26 | 2019-12-31 | 中海石油环保服务（天津）有限公司 | Culture medium and high-density fermentation method of petroleum hydrocarbon degrading bacteria |
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| CN111154702B (en) * | 2020-03-13 | 2023-07-28 | 辽宁大学 | Enterobacter cloacae for degrading petroleum pollution and application thereof |
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