CN108203701A - Complex microbial inoculum and its preparation method and application - Google Patents
Complex microbial inoculum and its preparation method and application Download PDFInfo
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- CN108203701A CN108203701A CN201611179047.4A CN201611179047A CN108203701A CN 108203701 A CN108203701 A CN 108203701A CN 201611179047 A CN201611179047 A CN 201611179047A CN 108203701 A CN108203701 A CN 108203701A
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- microbial inoculum
- hydrocarbon
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- pseudomonad
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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
- C02F3/343—Biological treatment of water, waste water, or sewage characterised by the microorganisms used for digestion of grease, fat, oil
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/32—Hydrocarbons, e.g. oil
-
- 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
Abstract
The invention discloses a kind of complex microbial inoculums and its preparation method and application, it is related to field of environment microorganism, the microorganism main body of complex microbial inoculum includes diesel oil alkane eating bacteria, sponge pseudomonad, Venice acinetobacter calcoaceticus and except hydrocarbon sea bacillus, this complex microbial inoculum can be applied to decomposing petroleum hydrocarbon, further apply the petroleum hydrocarbon in degradation sewage, it being capable of fully and effectively a large amount of existing straight-chain hydrocarbons in decomposing petroleum hydrocarbon, branched-chain hydrocarbons and aromatic hydrocarbon, and removal rate is high, its application in terms of decomposing petroleum hydrocarbon can efficiently solve the environmental problem of petroleum pollution in ocean.In addition, this complex microbial inoculum by diesel oil alkane eating bacteria, sponge pseudomonad, Venice acinetobacter calcoaceticus and except hydrocarbon sea bacillus be inoculated in microbial inoculum culture medium carry out co-incubation and obtain, preparation process is simple, device requirement is brief, operation is simple, the environmental condition needed is mild, suitable for commercial introduction and application.
Description
Technical field
The present invention relates to field of environment microorganism, in particular to a kind of complex microbial inoculum and preparation method thereof
And application.
Background technology
Oil pollution majority is happened in marine environment, ocean and surrounding ecological environment can be caused very serious
Harm, not only causes death of the large area marine site anoxic so as to cause marine flora and fauna, can also seabeach be caused to lie waste, and destroys marine products
Cultivation and Solar pond production damage coastal recreational area, reduce the service function and value of marine ecosystems；In addition, many oil
Hydro carbons is entered by food chain in fishes and shrimps body of shellfish, is influenced its economic value, and enter human body by food enrichment, is endangered the mankind
Health.Therefore, during oil pollution the marine eco-environment biggest threat.
Petroleum hydrocarbon ingredient is extremely complex, without all components in any microorganism energy degraded oil.Existing use
The microorganism of petroleum component in oily waste water of degrading is with removal efficiency is low, degradation aromatic hydrocarbon is not comprehensive, environmental suitability
With functional stabilization it is poor the shortcomings of.
Invention content
It, being capable of fully and effectively decomposing petroleum hydrocarbon simultaneously the purpose of the present invention is to provide a kind of complex microbial inoculum
In a large amount of existing straight-chain hydrocarbons, branched-chain hydrocarbons and aromatic hydrocarbon, the removal rate of the petroleum hydrocarbon in petroleum-polluted seawater can be improved,
So as to be conducive to the protection of marine environment.
It, can be same to obtain one kind another object of the present invention is to provide the preparation method of mentioned microorganism composite bacteria agent
When fully and effectively in decomposing petroleum hydrocarbon a large amount of existing straight-chain hydrocarbons, branched-chain hydrocarbons and aromatic hydrocarbon complex microbial inoculum, and should
Environmental condition needed for preparation method is mild and simple, convenient.
Another object of the present invention is to provide application of the mentioned microorganism composite bacteria agent in decomposing petroleum hydrocarbon, so that energy
Enough more effective, quick, comprehensively decomposing petroleum hydrocarbon.
Another object of the present invention is to provide the application of petroleum hydrocarbon of the mentioned microorganism composite bacteria agent in sewage of degrading,
With the petroleum hydrocarbon in the sewage that can fast and effeciently degrade, the removal rate of the petroleum hydrocarbon in petroleum-polluted sewage is improved, is reached
To preferable wastewater treatment efficiency.
The invention is realized in this way：
A kind of complex microbial inoculum, the microorganism main body of complex microbial inoculum include diesel oil alkane eating bacteria
(Alcanivorax dieselolei), sponge pseudomonad (Pseudomonas pachastrellae), Venice not lever
Bacterium (Acinetobacter venetianus) and except hydrocarbon sea bacillus (Marinobacter
hydrocarbonoclasticus)。
The preparation method of mentioned microorganism composite bacteria agent, including：By diesel oil alkane eating bacteria (Alcanivorax
Dieselolei), sponge pseudomonad (Pseudomonas pachastrellae), Venice acinetobacter calcoaceticus
(Acinetobacter venetianus) and except hydrocarbon sea bacillus (Marinobacter hydrocarbonoclasticus)
It is inoculated in microbial inoculum culture medium and carries out co-incubation, obtain complex microbial inoculum.
Mentioned microorganism composite bacteria agent is in the application of decomposing petroleum hydrocarbon.
The application of petroleum hydrocarbon of the mentioned microorganism composite bacteria agent in sewage of degrading.
Complex microbial inoculum of the embodiment of the present invention and preparation method thereof and its petroleum hydrocarbon in sewage of degrading
The advantageous effect of application is：Four kinds of microorganism main body diesel oil alkane eating bacterias, sponge pseudomonad, Vinnies in complex microbial inoculum
This acinetobacter calcoaceticus and except can mutually be cooperateed between the bacillus of hydrocarbon sea, and can mutually improve survival ability in the seawater, makes
Stability each other in the seawater is more preferable, and then reach can be more comprehensively efficiently a large amount of existing in decomposing petroleum hydrocarbon
The effect of straight-chain hydrocarbons, branched-chain hydrocarbons and aromatic hydrocarbon.In addition, the complex microbial inoculum preparation process is simple, device requirement is brief, behaviour
Make simply, the environmental condition needed is mild, suitable for commercial introduction and application.Complex microbial inoculum is in terms of decomposing petroleum hydrocarbon
Using the environmental problem that can efficiently solve petroleum pollution in ocean, promote China Petroleum industry development, green technology
It reforms, break industry technology barrier with important researching value and great market application prospect.
Description of the drawings
It in order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range, for those of ordinary skill in the art, without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is 13 kinds of straight chains in the oil-containing culture medium of diesel oil alkane eating bacteria seed liquor processing that the embodiment of the present invention 4 provides
The trend chart of 3 days of the relative surplus amount of hydrocarbon；
Fig. 2 be in the oil-containing culture medium of sponge pseudomonad seed liquor processing that the embodiment of the present invention 4 provides 13 kinds it is straight
The trend chart of 3 days of the relative surplus amount of chain hydrocarbon；
Fig. 3 is 13 kinds in the oil-containing culture medium of Venice acinetobacter calcoaceticus seed liquor processing that the embodiment of the present invention 4 provides
The trend chart of 4 days of the relative surplus amount of straight-chain hydrocarbons；
Fig. 4 is 13 kinds of straight chains in the oil-containing culture medium except the processing of hydrocarbon sea bacillus seed liquor that the embodiment of the present invention 4 provides
The trend chart of 4 days of the relative surplus amount of hydrocarbon；
Fig. 5 is 13 kinds of straight-chain hydrocarbons in the oil-containing culture medium of complex microbial inoculum processing that the embodiment of the present invention 4 provides
Relative surplus amount the trend chart of 3 days；
Fig. 6 is 2 kinds of branched-chain hydrocarbons in the oil-containing culture medium of diesel oil alkane eating bacteria seed liquor processing that the embodiment of the present invention 4 provides
Relative surplus amount the trend chart of 3 days；
Fig. 7 is 2 kinds of branches in the oil-containing culture medium of sponge pseudomonad seed liquor processing that the embodiment of the present invention 4 provides
The trend chart of 3 days of the relative surplus amount of hydrocarbon；
Fig. 8 is 2 kinds of branch in the oil-containing culture medium of Venice acinetobacter calcoaceticus seed liquor processing that the embodiment of the present invention 4 provides
The trend chart of 3 days of the relative surplus amount of chain hydrocarbon；
Fig. 9 is 2 kinds of branched-chain hydrocarbons in the oil-containing culture medium except the processing of hydrocarbon sea bacillus seed liquor that the embodiment of the present invention 4 provides
Relative surplus amount the trend chart of 3 days；
Figure 10 is 2 kinds of branched-chain hydrocarbons in the oil-containing culture medium of complex microbial inoculum processing that the embodiment of the present invention 4 provides
Relative surplus amount the trend chart of 3 days；
Figure 11 is 12 kinds of fragrance in the oil-containing culture medium of diesel oil alkane eating bacteria seed liquor processing that the embodiment of the present invention 4 provides
The trend chart of 3 days of the relative surplus amount of hydrocarbon；
Figure 12 is 12 kinds of virtues in the oil-containing culture medium of sponge pseudomonad seed liquor processing that the embodiment of the present invention 4 provides
The trend chart of 3 days of the relative surplus amount of fragrant hydrocarbon；
Figure 13 is 12 kinds in the oil-containing culture medium of Venice acinetobacter calcoaceticus seed liquor processing that the embodiment of the present invention 4 provides
The trend chart of 3 days of the relative surplus amount of aromatic hydrocarbon；
Figure 14 is 12 kinds of fragrance in the oil-containing culture medium except the processing of hydrocarbon sea bacillus seed liquor that the embodiment of the present invention 4 provides
The trend chart of 3 days of the relative surplus amount of hydrocarbon；
Figure 15 is 12 kinds of aromatic hydrocarbon in the oil-containing culture medium of complex microbial inoculum processing that the embodiment of the present invention 4 provides
Relative surplus amount the trend chart of 3 days.
Specific embodiment
Purpose, technical scheme and advantage to make the embodiment of the present invention are clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, builds according to normal condition or manufacturer
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Complex microbial inoculum of the embodiment of the present invention and preparation method thereof and its application are specifically described below.
The microorganism main body of complex microbial inoculum includes diesel oil alkane eating bacteria (Alcanivorax dieselolei), sponge
Pseudomonad (Pseudomonas pachastrellae), Venice acinetobacter calcoaceticus (Acinetobacter venetianus)
And except hydrocarbon sea bacillus (Marinobacter hydrocarbonoclasticus).
Wherein, diesel oil alkane eating bacteria (Alcanivorax dieselolei), sponge pseudomonad (Pseudomonas
Pachastrellae), Venice acinetobacter calcoaceticus (Acinetobacter venetianus) and except hydrocarbon sea bacillus
(Marinobacter hydrocarbonoclasticus) is taken at Chinese Sea Microbiological Culture Collection administrative center
(MCCC).The strain number of diesel oil alkane eating bacteria (Alcanivorax dieselolei) is 2216L-B-5, and preservation registration number is
1A00001；The strain number of sponge pseudomonad (Pseudomonas pachastrellae) is HLB AS-3, and preservation is registered
Number be 1A00102；The strain number of Venice acinetobacter calcoaceticus (Acinetobacter venetianus) be WP02421, preservation
Registration number is 1A00294；Except hydrocarbon sea bacillus (Marinobacter hydrocarbonoclasticus) bacterial strain marked as
DLFJ 7-4, preservation registration number 1A03971.
Diesel oil alkane eating bacteria, sponge pseudomonad, Venice acinetobacter calcoaceticus and the preservation except hydrocarbon sea bacillus can be by four
Kind bacterial strain, which is accessed in corresponding culture medium, is made bacterium solution and by the bacterium solution of each bacterial strain and 40% glycerine with 1：1-1：1.5 volume ratio
After being mixed, it is positioned over -70 DEG C of refrigerators.
Wherein, the culture medium of corresponding diesel oil alkane eating bacteria is No. 1001 culture mediums, including：Sodium acetate 0.5-1.5g/L, albumen
Peptone 8-12g/L, dusty yeast 1-3g/L, plain broth 0.1-0.8g/L, ammonium nitrate 0.1-0.3g/L, trisodium citrate 0.1-
0.8g/L, potassium dihydrogen phosphate 0.2-0.8g/L and artificial synthesized seawater, pH 7.0-7.8.
The sponge pseudomonad culture medium of corresponding sponge pseudomonad is that No. 0472 culture medium includes：Dusty yeast 3-7g/L,
Peptone 8-12g/L, sodium chloride 20-35g/L and deionized water；PH is 6.5-7.5.
Venice acinetobacter calcoaceticus culture medium of corresponding Venice acinetobacter calcoaceticus is that No. 0033 culture medium includes：Dusty yeast 3-
7g/L, peptone 8-12g/L, sodium chloride 5-15g/L；Solvent is deionized water, pH 6.5-7.5.
The corresponding hydrocarbon sea baccilus medium that removes except hydrocarbon sea bacillus is that No. 0821 culture medium includes：Sodium acetate 3-8g/L, albumen
Peptone 0.2-0.8g/L, dusty yeast 0.2-0.8g/L, glucose 0.2-0.8g/L, sucrose 0.2-0.8g/L, sodium citrate 0.02-
0.08g/L, DL-malic acid 0.02-0.08g/L, ammonium nitrate 0.8-1.5g/L, ammonium chloride 0.1-0.5g/L, potassium dihydrogen phosphate
0.2-0.8g/L and artificial synthesized seawater, pH 7.0-8.0.
Wherein, artificial synthesized seawater is：Sodium chloride 20-28g/L, six water magnesium sulfate 8-14g/L, sodium sulphate 3-5g/L, six
Water calcium chloride 1-3g/L, potassium chloride 0.5-1.0g/L, potassium bromide 0.05-0.2g/L, boric acid 0.01-0.05g/L, nine water sodium metasilicate
4-8mg/L, six water strontium chloride 0.02-0.08g/L, sodium fluoride 1-5mg/L, ammonium nitrate 1-6mg/L, ferric phosphate 0.5-1.5mg/L；
Solvent is deionized water；PH is 6.8-7.8.
In a preferred embodiment, the microorganism main body diesel oil alkane eating bacteria in complex microbial inoculum, sponge are false single
Born of the same parents bacterium, Venice acinetobacter calcoaceticus and exist in the form of bacterium solution except hydrocarbon sea bacillus is inoculated in microbial inoculum culture medium, and in microorganism
Concentration in composite bacteria agent is each independently 107-109cfu/mL.When diesel oil alkane eating bacteria, sponge pseudomonad, Venice are motionless
Bacillus and except concentration of the four kinds of bacterial strains of hydrocarbon sea bacillus in complex microbial inoculum is respectively positioned on 107-109Cfu/mL concentration ranges
When, it preferably can quickly and effectively be degraded, while the combination and compatibility between four kinds of bacterial strains to carrying out petroleum hydrocarbon, Neng Gouxiang
Coordinated is mutually carried out, improves the survival ability and activity of each bacterial strain, and then reach preferably degradation effect.It needs to illustrate
, in other embodiments, the microorganism main body diesel oil alkane eating bacteria in complex microbial inoculum, sponge pseudomonad, Vinnie
This acinetobacter calcoaceticus and except hydrocarbon sea bacillus can be existed in the form of non-bacterium solution, such as dry microbial inoculum.
When the Concentration portion of four kinds of bacterial strains or all less than 107-109During the concentration range of cfu/mL, the microorganism is compound
Microbial inoculum reduces the degradation effect and degradation rate of petroleum hydrocarbon, and the requirement of fast degradation is not achieved, and increases process costs.When four
Plant the Concentration portion of bacterial strain or all greater than 107-109During the concentration range of cfu/mL, complex microbial inoculum is in degraded oil
Excessive impurity can be introduced during hydrocarbon, is increased so as to cause the COD contents of the sewage containing petroleum hydrocarbon.
The preparation method of mentioned microorganism composite bacteria agent will be described further below, the preparation of complex microbial inoculum
Method includes：
S1, first time activation culture
Specifically, first time activation culture is by diesel oil alkane eating bacteria, sponge pseudomonad, Venice acinetobacter calcoaceticus and removes
Hydrocarbon sea bacillus, which is inoculated in respectively in activated strains fluid nutrient medium (liquid HLB), to be cultivated, will -70 DEG C preservation above-mentioned four
The glycerol tube strain of kind bacterial strain is inoculated in respectively in the 15mL culture tubes equipped with 2-5mL activated strains fluid nutrient mediums, will be cultivated
Pipe is placed in 20-28 DEG C, cultivates 48-120 hours in the oscillation shaking table of 180-220r/min.
Wherein, activated strains fluid nutrient medium includes tryptone 8-12g/L, yeast extract 4-6g/L and sodium chloride
25-35g/L and deionized water；PH value is 6-7.5.
S2, second of activation culture
Second of activation culture is will to be inoculated in activated strains solid respectively by the bacterium solution that first time activation culture obtains
It is cultivated in culture medium (solid HLB), the bacterium solution that first time activation culture obtains will be passed through and line activated strains solid
Rejuvenation culture 24-96h in 20-28 DEG C of constant incubator is placed in culture medium.
Wherein, activated strains solid medium includes tryptone 8-12g/L, yeast extract 4-6g/L, sodium chloride 5-
35g/L and agar powder 1.2-2.0g/L and deionized water；PH value is 6-7.5.
The activity of four kinds of bacterial strains and numerous is greatly increased by above-mentioned first time activation culture and second of activation culture
Grow ability so that it there can be better biological property.In addition, the activated strains fluid nutrient medium of above-mentioned selection and activation bacterium
The component and proportioning of strain solid medium, activation that can be preferably for four kinds of strains provides nutrition, to reach best work
Change effect.
S3, it is incubated overnight.
Specifically, the bacterium colony on the tablet after second of activation culture is respectively inoculated in equipped with 2-5mL liquid HLB's
15mL culture tubes are placed in 20-28 DEG C, are incubated overnight in the oscillation shaking table of 180-200r/min.Further bacterium solution is carried out to stay overnight training
Supporting enables bacterial strain to be properly arrived at logarithmic phase, so as to good biological property.
S4, seed liquor culture
By diesel oil alkane eating bacteria, sponge pseudomonad, Venice acinetobacter calcoaceticus and except hydrocarbon sea bacillus be inoculated in microbial inoculum training
Before supporting base progress co-incubation, four kinds of bacterium solutions after being first incubated overnight are inoculated in seed liquor with 1% inoculum concentration respectively
Culture medium carries out seed liquor culture, respectively obtains diesel oil alkane eating bacteria seed liquor, sponge pseudomonad seed liquor, Venice not lever
Bacterium seed liquor and except hydrocarbon sea bacillus seed liquor.Further, the starting pH of seed liquid culture medium is 6.0-8.0, is cultivated in 20-
28 DEG C, in the oscillation shaking table of 180-200r/min under conditions of carry out, incubation time 12-16h.The diesel oil that will be obtained after culture
Alkane eating bacteria seed liquor, sponge pseudomonad seed liquor, Venice acinetobacter calcoaceticus seed liquor and except hydrocarbon sea bacillus seed liquor exists
3-6 minutes are centrifuged under 5000-8000 revs/min, and collection diesel oil alkane eating bacteria thalline, sponge pseudomonad thalline, Venice are motionless respectively
Bacillus thalline and except hydrocarbon sea bacillus thalline.
Wherein, seed liquid culture medium includes trisodium citrate dihydrate 15-25g/L, ammonium chloride 0.8-1.5g/L, three hydration phosphorus
Sour hydrogen dipotassium 0.2-0.8g/L, potassium dihydrogen phosphate 0.1-0.4g/L and artificial synthesized seawater.It is artificial in seed liquid culture medium
The ingredient of synthetic seawater is identical with the ingredient of the artificial synthesized seawater in No. 0821 culture medium so that diesel oil alkane eating bacteria, sponge are false
Monad, Venice acinetobacter calcoaceticus and except hydrocarbon sea bacillus can more adapt to distinctive ocean ring in the culture of seed liquor
Border, the complex microbial inoculum being prepared finally to be enabled preferably to degrade the petroleum hydrocarbon in petroleum-polluted seawater.
Secondly, it by the diesel oil alkane eating bacteria thalline of acquisition, sponge pseudomonad thalline, Venice acinetobacter calcoaceticus thalline and removes
Hydrocarbon sea bacillus thalline is resuspended by 0.5-2% inoculum concentrations and is placed in 20-28 DEG C, the oscillation shaking table of 180-200r/min into microbial inoculum culture medium
Middle progress co-incubation 8-13h so that diesel oil alkane eating bacteria, sponge pseudomonad, Venice acinetobacter calcoaceticus and except hydrocarbon sea bacillus
Complex microbial inoculum concentration each 107-109In the range of cfu/mL.
Preferably, microbial inoculum culture medium includes trisodium citrate dihydrate 15-25g/L, ammonium chloride 0.8-1.5g/L, three hydration phosphorus
Sour hydrogen dipotassium 0.2-0.8g/L, potassium dihydrogen phosphate 0.1-0.4g/L and artificial synthesized seawater.Artificial conjunction in microbial inoculum culture medium
Ingredient into seawater is identical with the ingredient except the artificial synthesized seawater in the baccilus medium of hydrocarbon sea, so that diesel oil alkane eating bacteria,
Sponge pseudomonad, Venice acinetobacter calcoaceticus and except hydrocarbon sea bacillus can be grown in distinctive marine environment jointly,
The complex microbial inoculum being prepared finally to be enabled to better adapt to briny environment, so can preferably be directed to by
Petroleum hydrocarbon in the seawater of oil pollution is degraded.
It should be noted that the preparation method of complex microbial inoculum is not limited to above-mentioned steps, it can also be directly by bavin
Oily alkane eating bacteria, sponge pseudomonad, Venice acinetobacter calcoaceticus and except hydrocarbon sea bacillus without activation culture or is incubated overnight,
Also can achieve the effect that degrade to petroleum hydrocarbon to a certain extent.In the preparation process of mentioned microorganism composite bacteria agent,
The activation of bacterial strain and the culture of seed liquor etc. can carry out deleting and adjusting for step according to the actual needs.
The embodiment of the present invention additionally provides application of the mentioned microorganism composite bacteria agent in decomposing petroleum hydrocarbon.Mentioned microorganism is answered
Microbial inoculum is closed especially suitable for aromatic hydrocarbon, linear paraffin and the branched paraffin in decomposing petroleum hydrocarbon.
The embodiment of the present invention additionally provides the application of petroleum hydrocarbon of the complex microbial inoculum in sewage of degrading.Above-mentioned micro- life
Object composite bacteria agent is especially suitable for petroleum-polluted seawater of degrading.
The feature and performance of the present invention are described in further detail with reference to embodiments.
Embodiment 1
First, the preservation for being taken at Chinese Sea Microbiological Culture Collection administrative center (MCCC) of -70 DEG C of preservations is stepped on
Sponge pseudomonad that diesel oil alkane eating bacteria that mark is 1A00001, preservation registration number are 1A00102, preservation registration number are
Venice acinetobacter calcoaceticus and preservation registration number of 1A00294 is 1A03971 except the glycerol tube strain of hydrocarbon sea bacillus connects respectively
Kind in the 15mL culture tubes equipped with 5mL liquid HLB, culture tube is placed in 28 DEG C, cultivates 120 in the oscillation shaking table of 220r/min
Hour.Then, obtained bacterium solution is lined and rejuvenation culture in 28 DEG C of constant incubators is placed in activated strains solid medium
Bacterium colony on tablet is respectively inoculated in the 15mL culture tubes equipped with 5mL liquid HLB and is placed in 28 DEG C, the oscillation of 200r/min by 96h
It is incubated overnight in shaking table.
Secondly, the bacterium solution obtained after being incubated overnight is inoculated in starting pH as 8.0 seed using 1% inoculum concentration respectively
In liquid culture medium, it is placed in 28 DEG C, cultivates 16h in the oscillation shaking table of 200r/min, by each seed liquor obtained after culture 8000
It is centrifuged under rev/min 6 minutes and collects thalline.Then, by diesel oil alkane eating bacteria thalline, sponge pseudomonad thalline, Venice not lever
Bacterium thalline and except hydrocarbon sea bacillus thalline is resuspended by 2% inoculum concentration in microbial inoculum culture medium jointly, is placed in 28 DEG C, 200r/min
Oscillation shaking table in cultivate 13h, obtain complex microbial inoculum.In the complex microbial inoculum, the concentration of diesel oil alkane eating bacteria
8.765×108cfu/mL；The concentration 8.338 × 10 of sponge pseudomonad8cfu/mL；The concentration 8.956 of Venice acinetobacter calcoaceticus
×108cfu/mL；Except the concentration 8.114 × 10 of hydrocarbon sea bacillus8cfu/mL。
Wherein, liquid HLB includes tryptone 12g/L, yeast extract 6g/L and sodium chloride 35g/L and deionization
Water, pH value 7.5；Activated strains solid medium include tryptone 12g/L, yeast extract 6g/L, sodium chloride 35g/L and
Agar powder 2.0g/L and deionized water, pH value 7.5；Seed liquid culture medium includes trisodium citrate dihydrate 25g/L, ammonium chloride
1.5g/L, dipotassium hydrogen phosphate trihydrate 0.8g/L, potassium dihydrogen phosphate 0.4g/L and artificial synthesized seawater；Microbial inoculum culture medium includes
Trisodium citrate dihydrate 25g/L, ammonium chloride 1.4g/L, dipotassium hydrogen phosphate trihydrate 0.7g/L, potassium dihydrogen phosphate 0.4g/L and
Artificial synthesized seawater.
Further, artificial synthesized seawater is：Sodium chloride 28g/L, six water magnesium sulfate 14g/L, sodium sulphate 5g/L, six water chlorinations
Calcium 3g/L, potassium chloride 1.0g/L, potassium bromide 0.2g/L, boric acid 0.05g/L, nine water sodium metasilicate 8mg/L, six water strontium chloride 0.08g/
L, sodium fluoride 5mg/L, ammonium nitrate 6mg/L, ferric phosphate 1.5mg/L；Solvent is deionized water；PH is 7.8.
Embodiment 2
First, the preservation for being taken at Chinese Sea Microbiological Culture Collection administrative center (MCCC) of -70 DEG C of preservations is stepped on
Sponge pseudomonad that diesel oil alkane eating bacteria that mark is 1A00001, preservation registration number are 1A00102, preservation registration number are
Venice acinetobacter calcoaceticus and preservation registration number of 1A00294 is 1A03971 except the glycerol tube strain of hydrocarbon sea bacillus connects respectively
Kind in the 15mL culture tubes equipped with 2mL liquid HLB, culture tube is placed in 20 DEG C, cultivates 48 in the oscillation shaking table of 180r/min
Hour.Then, obtained bacterium solution is lined and rejuvenation culture in 20 DEG C of constant incubators is placed in activated strains solid medium
For 24 hours, the bacterium colony on tablet is respectively inoculated in the 15mL culture tubes equipped with 2mL liquid HLB and is placed in 20 DEG C, the oscillation of 180r/min
It is incubated overnight in shaking table.
Secondly, the bacterium solution obtained after being incubated overnight is inoculated in starting pH as 6.0 seed using 1% inoculum concentration respectively
In liquid culture medium, it is placed in 20 DEG C, cultivates 12h in the oscillation shaking table of 180r/min, by each seed liquor obtained after culture 5000
It is centrifuged under rev/min 3 minutes and collects thalline.Then, by diesel oil alkane eating bacteria thalline, sponge pseudomonad thalline, Venice not lever
Bacterium thalline and except hydrocarbon sea bacillus thalline is resuspended by 0.5% inoculum concentration in microbial inoculum culture medium jointly, is placed in 20 DEG C, 180r/
8h is cultivated in the oscillation shaking table of min, obtains complex microbial inoculum.In the complex microbial inoculum, the concentration of diesel oil alkane eating bacteria
7.765×107cfu/mL；The concentration 8.338 × 10 of sponge pseudomonad7cfu/mL；The concentration 5.954 of Venice acinetobacter calcoaceticus
×107cfu/mL；Except the concentration 3.513 × 10 of hydrocarbon sea bacillus7cfu/mL。
Wherein, liquid HLB includes tryptone 8g/L, yeast extract 4g/L and sodium chloride 25g/L and deionized water,
PH value is 6；Activated strains solid medium includes tryptone 8g/L, yeast extract 4g/L, sodium chloride 5g/L and agar powder
1.2g/L and deionized water, pH value 6；Seed liquid culture medium include trisodium citrate dihydrate 15g/L, ammonium chloride 0.8g/L,
Dipotassium hydrogen phosphate trihydrate 0.2g/L, potassium dihydrogen phosphate 0.1g/L and artificial synthesized seawater；Microbial inoculum culture medium includes two hydrations
Sodium citrate 15g/L, ammonium chloride 0.9g/L, dipotassium hydrogen phosphate trihydrate 0.3g/L, potassium dihydrogen phosphate 0.1g/L and artificial conjunction
Into seawater.
Further, artificial synthesized seawater is：Sodium chloride 20g/L, six water magnesium sulfate 8g/L, sodium sulphate 3g/L, six water chlorinations
Calcium 1g/L, potassium chloride 0.5-1.0g/L, potassium bromide 0.05-0.2g/L, boric acid 0.01g/L, nine water sodium metasilicate 4mg/L, six water chlorine
Change strontium 0.02g/L, sodium fluoride 1mg/L, ammonium nitrate 1mg/L, ferric phosphate 0.5mg/L；Solvent is deionized water；PH is 6.8.
Embodiment 3
First, the preservation for being taken at Chinese Sea Microbiological Culture Collection administrative center (MCCC) of -70 DEG C of preservations is stepped on
Sponge pseudomonad that diesel oil alkane eating bacteria that mark is 1A00001, preservation registration number are 1A00102, preservation registration number are
Venice acinetobacter calcoaceticus and preservation registration number of 1A00294 is 1A03971 except the glycerol tube strain of hydrocarbon sea bacillus connects respectively
Kind in the 15mL culture tubes equipped with 4mL liquid HLB, culture tube is placed in 25 DEG C, cultivates 72 in the oscillation shaking table of 200r/min
Hour.Then, obtained bacterium solution is lined and rejuvenation culture in 25 DEG C of constant incubators is placed in activated strains solid medium
Bacterium colony on tablet is respectively inoculated in the 15mL culture tubes equipped with 4mL liquid HLB and is placed in 25 DEG C, the oscillation of 190r/min by 48h
It is incubated overnight in shaking table.
Secondly, the bacterium solution obtained after being incubated overnight is inoculated in starting pH as 7.0 seed using 1% inoculum concentration respectively
In liquid culture medium, it is placed in 25 DEG C, middle culture 15h in the oscillation shaking table of 190r/min, each seed liquor obtained after culture is existed
It is centrifuged under 7000 revs/min 5 minutes and collects thalline.Then, by diesel oil alkane eating bacteria thalline, sponge pseudomonad thalline, Venice not
Lever bacterium thalline and except hydrocarbon sea bacillus thalline is resuspended by 1% inoculum concentration in microbial inoculum culture medium jointly, be placed in 25 DEG C,
11h is cultivated in the oscillation shaking table of 190r/min, obtains complex microbial inoculum.In the complex microbial inoculum, diesel oil alkane eating bacteria
Concentration 1.724 × 108cfu/mL；The concentration 8.988 × 10 of sponge pseudomonad7cfu/mL；The concentration of Venice acinetobacter calcoaceticus
2.954×108cfu/mL；Except the concentration 4.114 × 10 of hydrocarbon sea bacillus8cfu/mL。
Wherein, liquid HLB includes tryptone 10g/L, yeast extract 5g/L and sodium chloride 30g/L and deionization
Water, pH value 6.8；Activated strains solid medium include tryptone 10g/L, yeast extract 5g/L, sodium chloride 20g/L and
Agar powder 1.7g/L and deionized water, pH value 6.5；Seed liquid culture medium includes trisodium citrate dihydrate 20g/L, ammonium chloride
1.2g/L, dipotassium hydrogen phosphate trihydrate 0.6g/L, potassium dihydrogen phosphate 0.2g/L and artificial synthesized seawater；Microbial inoculum culture medium includes
Trisodium citrate dihydrate 21g/L, ammonium chloride 1.2g/L, dipotassium hydrogen phosphate trihydrate 0.5g/L, potassium dihydrogen phosphate 0.2g/L and
Artificial synthesized seawater.
Further, artificial synthesized seawater is：Sodium chloride 25g/L, six water magnesium sulfate 12g/L, sodium sulphate 4g/L, six water chlorinations
Calcium 2g/L, potassium chloride 0.8g/L, potassium bromide 0.1g/L, boric acid 0.03g/L, nine water sodium metasilicate 6mg/L, six water strontium chloride 0.05g/
L, sodium fluoride 4mg/L, ammonium nitrate 4mg/L, ferric phosphate 1.1mg/L；Solvent is deionized water；PH is 7.
Embodiment 4
First, the preservation for being taken at Chinese Sea Microbiological Culture Collection administrative center (MCCC) of -70 DEG C of preservations is stepped on
Sponge pseudomonad that diesel oil alkane eating bacteria that mark is 1A00001, preservation registration number are 1A00102, preservation registration number are
Venice acinetobacter calcoaceticus and preservation registration number of 1A00294 is 1A03971 except the glycerol tube strain of hydrocarbon sea bacillus connects respectively
Kind in the 15mL culture tubes equipped with 3mL liquid HLB, culture tube is placed in 24 DEG C, cultivates 100 in the oscillation shaking table of 190r/min
Hour.Then, obtained bacterium solution is lined and rejuvenation culture in 24 DEG C of constant incubators is placed in activated strains solid medium
Bacterium colony on tablet is respectively inoculated in the 15mL culture tubes equipped with 3mL liquid HLB and is placed in 26 DEG C, the oscillation of 188r/min by 68h
It is incubated overnight in shaking table.
Secondly, the bacterium solution obtained after being incubated overnight is inoculated in starting pH as 6.8 seed using 1% inoculum concentration respectively
In liquid culture medium, it is placed in 24 DEG C, cultivates 13h in the oscillation shaking table of 195r/min, by each seed liquor obtained after culture 6000
It is centrifuged under rev/min 5 minutes and collects thalline.Then, by diesel oil alkane eating bacteria thalline, sponge pseudomonad thalline, Venice not lever
Bacterium thalline and except hydrocarbon sea bacillus thalline is resuspended by 1.5% inoculum concentration in microbial inoculum culture medium jointly, is placed in 25 DEG C, 185r/
10h is cultivated in the oscillation shaking table of min, so as to obtain complex microbial inoculum.In the complex microbial inoculum, diesel oil alkane eating bacteria
Concentration 1.765 × 108cfu/mL；The concentration 9.318 × 10 of sponge pseudomonad7cfu/mL；The concentration of Venice acinetobacter calcoaceticus
2.954×108cfu/mL；Except the concentration 5.758 × 10 of hydrocarbon sea bacillus7cfu/mL。
Wherein, liquid HLB includes tryptone 10g/L, yeast extract 5g/L and sodium chloride 28g/L and deionization
Water, pH value 7.2；Activated strains solid medium include tryptone 9g/L, yeast extract 5g/L, sodium chloride 15g/L and
Agar powder 1.6g/L and deionized water, pH value 6.7；Seed liquid culture medium includes trisodium citrate dihydrate 19g/L, ammonium chloride
1.3g/L, dipotassium hydrogen phosphate trihydrate 0.5g/L, potassium dihydrogen phosphate 0.3g/L and artificial synthesized seawater；Microbial inoculum culture medium includes
Trisodium citrate dihydrate 18g/L, ammonium chloride 1.1g/L, dipotassium hydrogen phosphate trihydrate 0.7g/L, potassium dihydrogen phosphate 0.2g/L and
Artificial synthesized seawater.
Further, artificial synthesized seawater is：Sodium chloride 23g/L, six water magnesium sulfate 12g/L, sodium sulphate 4g/L, six water chlorinations
Calcium 2g/L, potassium chloride 0.8g/L, potassium bromide 0.13g/L, boric acid 0.02g/L, nine water sodium metasilicate 6mg/L, six water strontium chlorides
0.05g/L, sodium fluoride 3mg/L, ammonium nitrate 4mg/L, ferric phosphate 1.1mg/L；Solvent is deionized water；PH is 7.2.
Test example
Respectively by the seed liquor of each bacterial strain obtained in embodiment 4 and the complex microbial inoculum of acquisition respectively with 1%
Switching amount access microbial inoculum processing assay medium is placed in 20-28 DEG C, individually cultivates 3 days in the oscillation shaking table of 180-200r/min.
Since 0h, a sample is taken within every 24 hours, GC-MS is carried out after being extracted with organic solvent to the substance qualitative analysis that is degraded, and survey
Determine relative surplus oil mass, obtain degradation rate.Wherein GC-MS conditions are：Gas-chromatography (Agilent 7890A)；Mass detector
(Agilent HP5975C)；Chromatographic column is HP-5MS (30m × 0.25mm × 0.25 μm) capillary column；Injector temperature and detection
Temperature is 300,230 DEG C respectively, sample size 1ul, takes Splitless injecting samples, and auxiliary heating zone, level four bars temperature are respectively 280,
150 DEG C, carrier gas is nitrogen, flow velocity 1ml/min.Temperature program：60 DEG C of holding 1min, 300 DEG C are warming up to 5 DEG C/min speed
Keep 20min.MS uses full scan, and scanning range (m/z) is 40-600.
Wherein, microbial inoculum processing assay medium (referred to as D)：Ammonium chloride 1.2g/L, trisodium citrate dihydrate 4g/L, 0# bavin
Oil 1.1% (v/v), dipotassium hydrogen phosphate trihydrate 0.5g/L, potassium dihydrogen phosphate 0.3g/L and artificial synthesized seawater.Wherein, people
The ingredient of work synthetic seawater is identical with the ingredient of the artificial synthesized seawater in embodiment 3.
Qualitative point of the substance that is degraded in assay medium is handled by the GC-MS microbial inoculums handled above-mentioned 5 kinds of situations
Analysis, and measure in microbial inoculum processing assay medium and make Fig. 1-Figure 15 after relative surplus oil mass.
Fig. 1-Fig. 4 illustrate successively by embodiment 4 diesel oil alkane eating bacteria, sponge pseudomonad, Venice acinetobacter calcoaceticus,
Except the corresponding seed liquor of hydrocarbon sea bacillus individually handles the relative surplus amount of the microbial inoculum processing assay medium straight-chain hydrocarbons of 3 days of oil-containing
Variation tendency.Fig. 5 is illustrated the microbial inoculum processing experiment culture of the complex microbial inoculum obtained in embodiment 4 processing oil-containing
The variation tendency of the relative surplus amount of the base straight-chain hydrocarbons of 3 days.Wherein, the 1-13 in Fig. 1-Fig. 5 is that C11-C24 is free of the 13 of C23
Kind straight-chain hydrocarbons.
In the microbial inoculum processing assay medium that can be seen that the oil-containing by processing in three days referring to Fig. 1-Fig. 5, diesel oil alkane eating
Bacterium averagely reaches more than 80% to the removal rate of 13 kinds of straight-chain hydrocarbons；Sponge pseudomonad is to the removal rate average out to of 13 kinds of straight-chain hydrocarbons
To more than 85%；Venice acinetobacter calcoaceticus averagely reaches more than 90% to the removal rate of 13 kinds of straight-chain hydrocarbons, but connects after bacterium 24 to 13
The removal rate of kind straight-chain hydrocarbons is average less than 40%；Except hydrocarbon sea bacillus averagely reaches more than 80% to the removal rate of 13 kinds of straight-chain hydrocarbons；
Complex microbial inoculum averagely reaches more than 95% to the removal rate of 13 kinds of straight-chain hydrocarbons.With respect to four kinds bacterial strains act solely on oil
Hydrocarbon, complex microbial inoculum are more than the removal rate of 13 kinds of straight-chain hydrocarbons removal rate during four kinds of bacterial strain independent roles, and micro- life
Object composite bacteria agent averagely reaches more than 95% after bacterium is connect 24 hours to the removal rate of 13 kinds of straight-chain hydrocarbons, so as to microbial composite bacteria
When agent is with respect to four kinds of bacterial strain independent roles, to the degradation efficiency of straight-chain hydrocarbons faster.So as to which complex microbial inoculum is in degradation stone
In the application of petroleum hydrocarbon, it is used alone relative to four kinds of strains in decomposing petroleum hydrocarbon, in terms of the straight-chain hydrocarbons in decomposing petroleum hydrocarbon
With better removal rate and degradation efficiency, and then can be obtained in terms of petroleum hydrocarbon of the complex microbial inoculum in sewage of degrading
Application well, and be further applied in petroleum-polluted sewage.
Fig. 6-Fig. 9 illustrate successively by embodiment 4 diesel oil alkane eating bacteria, sponge pseudomonad, Venice acinetobacter calcoaceticus,
Except the corresponding seed liquor of hydrocarbon sea bacillus individually handles the relative surplus amount of the microbial inoculum processing assay medium branched-chain hydrocarbons of 3 days of oil-containing
Variation tendency.Figure 10 is illustrated the microbial inoculum processing experiment training of the complex microbial inoculum obtained in embodiment 4 processing oil-containing
Support the variation tendency of the relative surplus amount of the base branched-chain hydrocarbons of 3 days.1 and 2 in Fig. 6-Figure 10 be respectively two kinds of branch of C15 and C19
Chain hydrocarbon.
In the microbial inoculum processing assay medium that can be seen that the oil-containing by processing in three days referring to Fig. 6-Figure 10, diesel oil food
Alkane bacterium averagely reaches more than 80% to the removal rate of 2 kinds of branched-chain hydrocarbons；Sponge pseudomonad is to the removal rate average out to of 2 kinds of branched-chain hydrocarbons
To more than 90%；Venice acinetobacter calcoaceticus averagely reaches more than 90% to the removal rate of 2 kinds of branched-chain hydrocarbons；Except hydrocarbon sea bacillus is to 2 kinds
The removal rate of branched-chain hydrocarbons averagely reaches more than 80%；Complex microbial inoculum averagely reaches 95% to the removal rate of 2 kinds of branched-chain hydrocarbons
More than.With respect to four kinds bacterial strains act solely on petroleum hydrocarbon, and complex microbial inoculum is more than four kinds of bacterium to the removal rate of 2 kinds of branched-chain hydrocarbons
Removal rate during strain independent role, and complex microbial inoculum is averaged to the removal rate of 2 kinds of branched-chain hydrocarbons after bacterium is connect 24 hours
Reach more than 95%, thus when complex microbial inoculum is with respect to four kinds of bacterial strain independent roles, to the degradation efficiency of branched-chain hydrocarbons
Faster.So as to which complex microbial inoculum is in the application of decomposing petroleum hydrocarbon, it is used alone relative to four kinds of strains in degraded oil
During hydrocarbon, there is better removal rate and degradation efficiency, and then complex microbial inoculum in terms of the branched-chain hydrocarbons in decomposing petroleum hydrocarbon
It can be applied well in terms of petroleum hydrocarbon in sewage of degrading, and be further applied to petroleum-polluted sewage
In.
Figure 11 Figure 14 illustrate successively by embodiment 4 diesel oil alkane eating bacteria, sponge pseudomonad, Venice not lever
Bacterium, individually handled except the corresponding seed liquor of hydrocarbon sea bacillus oil-containing the microbial inoculum processing assay medium aromatic hydrocarbon of 3 days it is relatively surplus
The variation tendency of surplus.It is real that Figure 15 illustrates that the microbial inoculum by the complex microbial inoculum obtained in embodiment 4 processing oil-containing is handled
Test the variation tendency of the relative surplus amount of the culture medium aromatic hydrocarbon of 3 days.9 kinds of fragrance that 1-12 in Figure 11-Figure 15 is C9-C12
Hydrocarbon.
In the microbial inoculum processing assay medium that can be seen that the oil-containing by processing in three days referring to Figure 11-Figure 15, diesel oil food
Alkane bacterium averagely reaches more than 80% to the removal rate of 12 kinds of aromatic hydrocarbon；Sponge pseudomonad is averaged to the removal rate of 12 kinds of aromatic hydrocarbon
Reach more than 80%；Venice acinetobacter calcoaceticus finally averagely reaches more than 82% to the removal rate of 12 kinds of aromatic hydrocarbon, but connects bacterium
It is only average less than 20% to the removal rate of 12 kinds of aromatic hydrocarbon after 24 hours；Except hydrocarbon sea bacillus is averaged to the removal rate of 12 kinds of aromatic hydrocarbon
Reach more than 80%, but it is average less than 70% to the removal rate of 12 kinds of aromatic hydrocarbon after 48 hours to connect bacterium；Complex microbial inoculum pair
The removal rate of 12 kinds of aromatic hydrocarbon averagely reaches more than 98%.With respect to four kinds bacterial strains act solely on petroleum hydrocarbon, microbial composite bacteria
Agent is far longer than the removal rate of 12 kinds of aromatic hydrocarbon removal rate during four kinds of bacterial strain independent roles, and complex microbial inoculum exists
It connects bacterium and more than 95% is averagely reached to the removal rate of 2 kinds of aromatic hydrocarbon after 24 hours, so as to respect to four kinds bacterium of complex microbial inoculum
During strain independent role, to the degradation efficiency of aromatic hydrocarbon also faster.So as to which complex microbial inoculum is in the application of decomposing petroleum hydrocarbon
On, it is used alone in decomposing petroleum hydrocarbon, has in terms of the aromatic hydrocarbon in decomposing petroleum hydrocarbon better relative to four kinds of strains
Removal rate and degradation efficiency, and then can obtain well should in terms of petroleum hydrocarbon of the complex microbial inoculum in sewage of degrading
With, and be further applied in petroleum-polluted sewage.
It can obtain from the above analysis, the various hydro carbons effects of complex microbial inoculum processing are better than any single strain, to chain
The removal rate of hydrocarbon is up to more than 98%, and degradation efficiency is significantly larger than single strain up to more than 95%, to the removal of aromatic hydrocarbon
Degradation efficiency during effect.So as to which complex microbial inoculum is in the application of decomposing petroleum hydrocarbon, individually should relative to four kinds of strains
During used in decomposing petroleum hydrocarbon, better removal rate is respectively provided in terms of the chain hydrocarbon and aromatic hydrocarbon in decomposing petroleum hydrocarbon and degradation is imitated
Rate, and then can be applied well in terms of petroleum hydrocarbon of the complex microbial inoculum in sewage of degrading, and further should
For in petroleum-polluted sewage.
In conclusion four kinds of microorganism main body diesel oil alkane eating bacterias, sponge pseudomonad, Vinnies in complex microbial inoculum
This acinetobacter calcoaceticus and except can mutually be cooperateed between the bacillus of hydrocarbon sea, and can mutually improve survival ability in the seawater, makes
Stability each other in the seawater is more preferable, and then reach can be more comprehensively efficiently a large amount of existing in decomposing petroleum hydrocarbon
The effect of straight-chain hydrocarbons, branched-chain hydrocarbons and aromatic hydrocarbon.In addition, the microbial bacterial agent preparation process is simple, device requirement is brief, operation letter
Easily, the environmental condition needed is mild, suitable for commercial introduction and application；Its application in terms of decomposing petroleum hydrocarbon can be solved effectively
The certainly environmental problem of petroleum pollution in ocean is promoting the development of China Petroleum industry, the innovation of green technology, is breaking industry technology
Barrier has important researching value and great market application prospect.
Embodiments described above is part of the embodiment of the present invention, instead of all the embodiments.The reality of the present invention
The detailed description for applying example is not intended to limit the range of claimed invention, but is merely representative of the selected implementation of the present invention
Example.Based on the embodiments of the present invention, those of ordinary skill in the art are obtained without creative efforts
Every other embodiment, shall fall within the protection scope of the present invention.
Claims (14)
1. a kind of complex microbial inoculum, which is characterized in that the microorganism main body of the complex microbial inoculum is eaten including diesel oil
Alkane bacterium (Alcanivorax dieselolei), sponge pseudomonad (Pseudomonas pachastrellae), Venice are not
Lever bacterium (Acinetobacter venetianus) and except hydrocarbon sea bacillus (Marinobacter
hydrocarbonoclasticus)。
2. complex microbial inoculum according to claim 1, which is characterized in that the diesel oil alkane eating bacteria (Alcanivorax
Dieselolei), the sponge pseudomonad (Pseudomonas pachastrellae), Venice acinetobacter calcoaceticus
It is (Acinetobacter venetianus) and described except hydrocarbon sea bacillus (Marinobacter
Hydrocarbonoclasticus) concentration in the complex microbial inoculum is each independently 107-109cfu/mL。
3. complex microbial inoculum according to claim 1, which is characterized in that the diesel oil alkane eating bacteria (Alcanivorax
Dieselolei preservation registration number) be 1A00001, the sponge pseudomonad (Pseudomonas pachastrellae)
Preservation registration number for 1A00102, the preservation of Venice acinetobacter calcoaceticus (Acinetobacter venetianus) is registered
Number for 1A00294, the preservation registration number except hydrocarbon sea bacillus (Marinobacter hydrocarbonoclasticus) is
1A03971, depositary institution are Chinese Sea Microbiological Culture Collection administrative center.
4. a kind of preparation method of complex microbial inoculum as described in any one of claims 1 to 3, which is characterized in that it is wrapped
It includes：
By the diesel oil alkane eating bacteria (Alcanivorax dieselolei), the sponge pseudomonad (Pseudomonas
Pachastrellae), Venice acinetobacter calcoaceticus (Acinetobacter venetianus) and it is described remove hydrocarbon sea bacillus
(Marinobacter hydrocarbonoclasticus) is inoculated in microbial inoculum culture medium and carries out co-incubation, obtains micro- life
Object composite bacteria agent.
5. preparation method according to claim 4, which is characterized in that by the diesel oil alkane eating bacteria (Alcanivorax
Dieselolei), the sponge pseudomonad (Pseudomonas pachastrellae), Venice acinetobacter calcoaceticus
It is (Acinetobacter venetianus) and described except hydrocarbon sea bacillus (Marinobacter
Hydrocarbonoclasticus it is first inoculated in seed liquid culture medium and carries out respectively before) being inoculated in the microbial inoculum culture medium
Seed liquor culture, the seed liquid culture medium include trisodium citrate dihydrate 15-25g/L, ammonium chloride 0.8-1.5g/L, three hydrations
Dipotassium hydrogen phosphate 0.2-0.8g/L, potassium dihydrogen phosphate 0.1-0.4g/L and artificial synthesized seawater.
6. preparation method according to claim 5, which is characterized in that the starting pH of the seed liquid culture medium is 6.0-
8.0, it cultivates and is carried out under the conditions of 20-28 DEG C.
7. preparation method according to claim 5, which is characterized in that by the diesel oil alkane eating bacteria (Alcanivorax
Dieselolei), the sponge pseudomonad (Pseudomonas pachastrellae), Venice acinetobacter calcoaceticus
It is (Acinetobacter venetianus) and described except hydrocarbon sea bacillus (Marinobacter
Hydrocarbonoclasticus it is first inoculated in activated strains fluid nutrient medium before) being inoculated in the seed liquid culture medium
First time activation culture is carried out, the activated strains fluid nutrient medium includes tryptone 8-12g/L, yeast extract 4-6g/
L, sodium chloride 25-35g/L and deionized water；Wherein, pH value 6-7.5.
8. preparation method according to claim 7, which is characterized in that the bacterium that will be obtained by the first time activation culture
Liquid, which is inoculated in respectively in activated strains solid medium, carries out second of activation culture, and the activated strains solid medium includes
Tryptone 8-12g/L, yeast extract 4-6g/L, sodium chloride 5-35g/L and agar powder 1.2-2.0g/L and deionized water；
Wherein pH value is 6-7.5.
9. preparation method according to claim 4, which is characterized in that the microbial inoculum culture medium includes trisodium citrate dihydrate
15-25g/L, ammonium chloride 0.8-1.5g/L, dipotassium hydrogen phosphate trihydrate 0.2-0.8g/L, potassium dihydrogen phosphate 0.1-0.4g/L and
Artificial synthesized seawater.
10. the preparation method according to claim 5 or 9, which is characterized in that the artificial synthesized seawater includes：Sodium chloride
20-28g/L, six water magnesium sulfate 8-14g/L, sodium sulphate 3-5g/L, calcium chloride hexahydrate 1-3g/L, potassium chloride 0.5-1.0g/L, bromine
Change potassium 0.05-0.2g/L, boric acid 0.01-0.05g/L, nine water sodium metasilicate 4-8mg/L, six water strontium chloride 0.02-0.08g/L, fluorine
Change sodium 1-5mg/L, ammonium nitrate 1-6mg/L, ferric phosphate 0.5-1.5mg/L and deionized water, pH value 6.8-7.8.
11. complex microbial inoculum according to any one of claims 1 to 3 is in the application of decomposing petroleum hydrocarbon.
12. application according to claim 11, which is characterized in that the petroleum hydrocarbon includes aromatic hydrocarbon, straight-chain hydrocarbons and branch
Hydrocarbon.
13. the application of petroleum hydrocarbon of the complex microbial inoculum according to any one of claims 1 to 3 in sewage of degrading.
14. application according to claim 13, which is characterized in that the sewage is petroleum-polluted seawater.
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