CN106282051A - A kind of microbial bacterial agent preparation method of petroleum pollution in ocean degraded - Google Patents
A kind of microbial bacterial agent preparation method of petroleum pollution in ocean degraded Download PDFInfo
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- CN106282051A CN106282051A CN201610645599.3A CN201610645599A CN106282051A CN 106282051 A CN106282051 A CN 106282051A CN 201610645599 A CN201610645599 A CN 201610645599A CN 106282051 A CN106282051 A CN 106282051A
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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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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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
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/08—Seawater, e.g. for desalination
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Abstract
The invention belongs to marine pollution degraded field, particularly relate to the microbial bacterial agent preparation method of a kind of petroleum pollution in ocean degraded.After functional microorganism bulk fermentation is cultivated by the present invention; microorganism fungus kind three grade fermemtation form is used to spread cultivation bacterium solution; and use active zeolite absorption and wrap up with PGA; form microbial inoculum product; zeolite surface parcel protective agent (nutrient); the loss of microorganism in lower zeolite pore footpath as far as possible in infall process; microorganism can also be made as nutrient source using coating agent simultaneously; water body is not caused secondary pollution, thus reaches microbial inoculum living bacteria count ＞ 100,000,000/g and launch process lose the purpose that bacterium number is minimum.
Description
Technical field
The invention belongs to marine pollution degraded field, particularly relate to the microbial bacterial agent of a kind of petroleum pollution in ocean degraded
Preparation method.
Background technology
Offshore oil oil spilling and oil pollution problem always affect the major issue of marine environment and marine ecology.At present
For petroleum pollution in ocean, the particularly emergent management of Accidental Oil Spill accident, mainly for the ocean table of oil spilling early period of origination
Layer sea water, uses the modes such as physics interception, recovery to carry out emergency disposal.But, sink for seabed oil spilling or with water sports
The oil pollution being down in bottom sediment, there is presently no good physics, chemical method can process, oil in deposit
The environmental self-purification of degrading microorganism and artificial importing oil degradation microorganism are the masters solving marine sediment oil pollution at present
Want mode.
But, aforesaid way suffers a disadvantage in that 1, physics and chemical restoration, easily causes secondary pollution；2, repair
Multiple major part can only carry out enclosing of surface flotation light oil and block and clear up；3, recovery technique can only act on the short time, it is impossible to is formed
Long-lasting.
Summary of the invention
The present invention is directed to the deficiency that above-mentioned prior art exists, it is provided that the microbial bacteria of a kind of petroleum pollution in ocean degraded
Agent preparation method.
The technical scheme is that the microbial bacteria that a kind of petroleum pollution in ocean is degraded
Agent preparation method, comprises the following steps:
(1) seed liquor is prepared
A, preparation culture medium: in 950ml deionized water, add tryptone 10g, yeast extract 5g and sodium chloride
10g, shakes container until solute dissolves, adjusts pH to 7.0 by the NaOH solution of 5mol/L, then be settled to 1L with deionized water,
Steam sterilization 21min under 15ps i high pressure；
B, repair class strain in the inoculation of medium ocean of step a, and be placed on shaking table to cultivate to absorbance and be more than
1.0, microscopy prepares seed liquor without miscellaneous bacteria；
(2) bacterium solution is prepared
A, one grade fermemtation: in one grade fermemtation tank, by tryptone 450g, yeast leaching powder 120g, calcium chloride 1.4g, sulphuric acid
Magnesium 14g, sodium chloride 800g, ferrous sulfate 4g and hydrogen sulfate dipotassium 100g join in 80L water after stirring and dissolving, and regulation pH is extremely
7.5,121 DEG C, sterilizing under the conditions of 1.5MPa, it is cooled to 30 DEG C, seed liquor is aseptically seeded to one grade fermemtation tank；
B, second order fermentation: in second order fermentation tank, by tryptone 4800g, yeast leaching powder 1300g, calcium chloride 15g, sulfur
Acid magnesium 140g, sodium chloride 8000g, ferrous sulfate 40g and hydrogen sulfate dipotassium 1000g join in 800L water after stirring and dissolving, adjust
Joint pH to 7.5,121 DEG C, sterilizing under the conditions of 1.5MPa, is cooled to 30 DEG C；When in one grade fermemtation tank bacterium solution absorbance big
In 1.0 time, being aseptically transferred to by fermentation liquid in second order fermentation tank, aseptic filtration ventilation stir culture, multiple spot is aseptic
Sampling, monitors to absorbance more than 1.1, and miscellaneous bacteria rate controls, at below 10wt%, to complete second order fermentation；
C, three grade fermemtation: in three grade fermemtation tank, by tryptone 25kg, yeast leaching powder 7kg, calcium chloride 80g, magnesium sulfate
750g, sodium chloride 40kg, ferrous sulfate 210g and hydrogen sulfate dipotassium 5kg join in 4000L water after stirring and dissolving, and regulation pH is extremely
7.5,121 DEG C, sterilizing under the conditions of 1.5MPa, it is cooled to 30 DEG C, the fermentation liquid in second order fermentation tank is transferred to three grade fermemtation
Tank, aseptic filtration ventilation stir culture, multiple spot sterile sampling, monitor to absorbance more than 1.2, miscellaneous bacteria rate control 20wt% with
Under, complete three grade fermemtation；
D, preservation: by complete the zymocyte liquid of three grade fermemtation under the conditions of 4 DEG C stored under refrigeration in bacterium solution material feeding jar；
(3) absorption: the bacterium solution in bacterium solution material feeding jar is added stirring with the zeolite in zeolite material feeding jar according to weight ratio 2:1
In tank I, carry out Stirring, mixing and absorption 20min；
(4) control liquid: the zeolite after step (3) absorption microbial inoculum is transported on dewatering screen, by vibration, liquid is filtered, control
Liquid 6min；
(5) parcel: by concentration 7wt% poly-in product and the polyglutamic acid solution tank after step (4) mixing control fluid dewatering
Glutamic acid solution is simultaneously introduced in agitator tank II and is stirred, mixes, wraps up 10min；
(6) preserve: the microbial inoculum after step (5) being wrapped up loads preservation in jumbo bag.
Wherein, class strain is repaired in the ocean described in step (1) is bacillus cereus or sea Zymomonas mobilis.
Microbial inoculum weight in step (6) jumbo bag is 1 ± 0.1 ton, living bacteria count >=100,000,000/gram.
The invention has the beneficial effects as follows: after functional microorganism bulk fermentation is cultivated by the present invention, use microorganism fungus kind three
Level fermentation form spreads cultivation bacterium solution, and uses active zeolite absorption and wrap up with PGA, forms microbial inoculum product, and zeolite surface parcel is protected
Protecting agent (nutrient), in infall process, the loss of microorganism in lower zeolite pore footpath as far as possible, can also make microorganism simultaneously
Using coating agent as nutrient source, water body is not caused secondary pollution, thus reaches microbial inoculum living bacteria count ＞ 100,000,000/g and throw in
During lose the purpose that bacterium number is minimum.
Accompanying drawing explanation
Fig. 1 is the process chart of the present invention；
In figure, 1, zeolite material feeding jar；2, bacterium solution material feeding jar；3, agitator tank I；4, dewatering screen；5, agitator tank II；6, polyglutamic
Acid solution tank.
Detailed description of the invention
Being described principle and the feature of the present invention below in conjunction with example, example is served only for explaining the present invention, and
Non-for limiting the scope of the present invention.
Embodiment 1
The microbial bacterial agent preparation method of a kind of petroleum pollution in ocean degraded, comprises the following steps:
(1) seed liquor is prepared
A, preparation culture medium: in 950ml deionized water, add tryptone 10g, yeast extract 5g and sodium chloride
10g, shakes container until solute dissolves, adjusts pH to 7.0 by the NaOH solution of 5mol/L, then be settled to 1L with deionized water,
Steam sterilization 21min under 15ps i high pressure；
B, repair class strain in the inoculation of medium ocean of step a, and be placed on shaking table to cultivate to absorbance and be more than
1.0, microscopy prepares seed liquor without miscellaneous bacteria；
(2) bacterium solution is prepared
A, one grade fermemtation: in one grade fermemtation tank, by tryptone 450g, yeast leaching powder 120g, calcium chloride 1.4g, sulphuric acid
Magnesium 14g, sodium chloride 800g, ferrous sulfate 4g and hydrogen sulfate dipotassium 100g join in 80L water after stirring and dissolving, and regulation pH is extremely
7.5,121 DEG C, sterilizing under the conditions of 1.5MPa, it is cooled to 30 DEG C, seed liquor is aseptically seeded to one grade fermemtation tank；
B, second order fermentation: in second order fermentation tank, by tryptone 4800g, yeast leaching powder 1300g, calcium chloride 15g, sulfur
Acid magnesium 140g, sodium chloride 8000g, ferrous sulfate 40g and hydrogen sulfate dipotassium 1000g join in 800L water after stirring and dissolving, adjust
Joint pH to 7.5,121 DEG C, sterilizing under the conditions of 1.5MPa, is cooled to 30 DEG C；When in one grade fermemtation tank bacterium solution absorbance big
In 1.0 time, being aseptically transferred to by fermentation liquid in second order fermentation tank, aseptic filtration ventilation stir culture, multiple spot is aseptic
Sampling, monitors to absorbance more than 1.1, and miscellaneous bacteria rate controls, at below 10wt%, to complete second order fermentation；
C, three grade fermemtation: in three grade fermemtation tank, by tryptone 25kg, yeast leaching powder 7kg, calcium chloride 80g, magnesium sulfate
750g, sodium chloride 40kg, ferrous sulfate 210g and hydrogen sulfate dipotassium 5kg join in 4000L water after stirring and dissolving, and regulation pH is extremely
7.5,121 DEG C, sterilizing under the conditions of 1.5MPa, it is cooled to 30 DEG C, the fermentation liquid in second order fermentation tank is transferred to three grade fermemtation
Tank, aseptic filtration ventilation stir culture, multiple spot sterile sampling, monitor to absorbance more than 1.2, miscellaneous bacteria rate control 20wt% with
Under, complete three grade fermemtation；
D, preservation: by complete the zymocyte liquid of three grade fermemtation under the conditions of 4 DEG C stored under refrigeration in bacterium solution material feeding jar；
(3) absorption: the bacterium solution in bacterium solution material feeding jar is added stirring with the zeolite in zeolite material feeding jar according to weight ratio 2:1
In tank I, carry out Stirring, mixing and absorption 20min；
(4) control liquid: the zeolite after step (3) absorption microbial inoculum is transported on dewatering screen, by vibration, liquid is filtered, control
Liquid 6min；
(5) parcel: by concentration 7wt% poly-in product and the polyglutamic acid solution tank after step (4) mixing control fluid dewatering
Glutamic acid solution is simultaneously introduced in agitator tank II and is stirred, mixes, wraps up 10min；
(6) preserve: the microbial inoculum after step (5) being wrapped up loads preservation in jumbo bag.
After testing, the microbial inoculum weight in every jumbo bag is 1 ± 0.1 ton, living bacteria count >=100,000,000/gram.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all spirit in the present invention and
Within principle, any modification, equivalent substitution and improvement etc. made, should be included within the scope of the present invention.
Claims (3)
1. the microbial bacterial agent preparation method of a petroleum pollution in ocean degraded, it is characterised in that comprise the following steps:
(1) seed liquor is prepared
A, preparation culture medium: in 950ml deionized water, add tryptone 10g, yeast extract 5g and sodium chloride 10g, shake
Visibly moved device, until solute dissolves, is adjusted pH to 7.0 by the NaOH solution of 5mol/L, then is settled to 1L with deionized water, high at 15psi
Pressure steam sterilization 21min；
B, repair class strain in the inoculation of medium ocean of step a, and be placed on shaking table and cultivate to absorbance more than 1.0,
Microscopy prepares seed liquor without miscellaneous bacteria；
(2) bacterium solution is prepared
A, one grade fermemtation: in one grade fermemtation tank, by tryptone 450g, yeast leaching powder 120g, calcium chloride 1.4g, magnesium sulfate
14g, sodium chloride 800g, ferrous sulfate 4g and hydrogen sulfate dipotassium 100g join in 80L water after stirring and dissolving, regulate pH to 7.5,
121 DEG C, sterilizing under the conditions of 1.5MPa, it is cooled to 30 DEG C, seed liquor is aseptically seeded to one grade fermemtation tank；
B, second order fermentation: in second order fermentation tank, by tryptone 4800g, yeast leaching powder 1300g, calcium chloride 15g, magnesium sulfate
140g, sodium chloride 8000g, ferrous sulfate 40g and hydrogen sulfate dipotassium 1000g join in 800L water after stirring and dissolving, regulate pH
To 7.5,121 DEG C, sterilizing under the conditions of 1.5MPa, it is cooled to 30 DEG C；When in one grade fermemtation tank bacterium solution absorbance more than 1.0
Time, fermentation liquid is aseptically transferred in second order fermentation tank, aseptic filtration ventilation stir culture, multiple spot sterile sampling,
Monitoring to absorbance more than 1.1, miscellaneous bacteria rate controls, at below 10wt%, to complete second order fermentation；
C, three grade fermemtation: in three grade fermemtation tank, by tryptone 25kg, yeast leaching powder 7kg, calcium chloride 80g, magnesium sulfate
750g, sodium chloride 40kg, ferrous sulfate 210g and hydrogen sulfate dipotassium 5kg join in 4000L water after stirring and dissolving, and regulation pH is extremely
7.5,121 DEG C, sterilizing under the conditions of 1.5MPa, it is cooled to 30 DEG C, the fermentation liquid in second order fermentation tank is transferred to three grade fermemtation
Tank, aseptic filtration ventilation stir culture, multiple spot sterile sampling, monitor to absorbance more than 1.2, miscellaneous bacteria rate control 20wt% with
Under, complete three grade fermemtation；
D, preservation: by complete the zymocyte liquid of three grade fermemtation under the conditions of 4 DEG C stored under refrigeration in bacterium solution material feeding jar；
(3) absorption: the bacterium solution in bacterium solution material feeding jar is added agitator tank I with the zeolite in zeolite material feeding jar according to weight ratio 2:1
In, carry out Stirring, mixing and absorption 20min；
(4) control liquid: the zeolite after step (3) absorption microbial inoculum is transported on dewatering screen, by vibration, liquid is filtered, control liquid
6min；
(5) parcel: by the product after step (4) mixing control fluid dewatering and the polyglutamic of concentration 7wt% in polyglutamic acid solution tank
Acid solution is simultaneously introduced in agitator tank II and is stirred, mixes, wraps up 10min；
(6) preserve: the microbial inoculum after step (5) being wrapped up loads preservation in jumbo bag.
Preparation method the most according to claim 1, it is characterised in that the ocean described in step (1) is repaired class strain and is
Bacillus cereus or sea Zymomonas mobilis.
Preparation method the most according to claim 1, it is characterised in that the microbial inoculum weight in step (6) jumbo bag is 1 ± 0.1
Ton, living bacteria count >=100,000,000/gram.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610645599.3A CN106282051A (en) | 2016-08-08 | 2016-08-08 | A kind of microbial bacterial agent preparation method of petroleum pollution in ocean degraded |
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| CN201610645599.3A CN106282051A (en) | 2016-08-08 | 2016-08-08 | A kind of microbial bacterial agent preparation method of petroleum pollution in ocean degraded |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101050435A (en) * | 2007-03-16 | 2007-10-10 | 山东省科学院生物研究所 | Solid microbe agent for degrading petroleum pollution, and petroleum products, and preparation method |
| CN101597576A (en) * | 2009-06-30 | 2009-12-09 | 山东省科学院生物研究所 | A kind of solid microbial agent to remedy soil contaminated by petroleum and its production and application |
| CN102839137A (en) * | 2012-07-27 | 2012-12-26 | 长安大学 | Solid microbial bacterium agent for petroleum degradation and preparation method thereof |
| CN103045579A (en) * | 2012-12-17 | 2013-04-17 | 南开大学 | Microbial remediation curing adsorbing bacterial preparation applicable to marine environment petroleum pollution as well as preparation method and application of same |
-
2016
- 2016-08-08 CN CN201610645599.3A patent/CN106282051A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101050435A (en) * | 2007-03-16 | 2007-10-10 | 山东省科学院生物研究所 | Solid microbe agent for degrading petroleum pollution, and petroleum products, and preparation method |
| CN101597576A (en) * | 2009-06-30 | 2009-12-09 | 山东省科学院生物研究所 | A kind of solid microbial agent to remedy soil contaminated by petroleum and its production and application |
| CN102839137A (en) * | 2012-07-27 | 2012-12-26 | 长安大学 | Solid microbial bacterium agent for petroleum degradation and preparation method thereof |
| CN103045579A (en) * | 2012-12-17 | 2013-04-17 | 南开大学 | Microbial remediation curing adsorbing bacterial preparation applicable to marine environment petroleum pollution as well as preparation method and application of same |
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Application publication date: 20170104 |
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