CN107058104A - The cultural method of oil degradation bacteria - Google Patents
The cultural method of oil degradation bacteria Download PDFInfo
- Publication number
- CN107058104A CN107058104A CN201710248046.9A CN201710248046A CN107058104A CN 107058104 A CN107058104 A CN 107058104A CN 201710248046 A CN201710248046 A CN 201710248046A CN 107058104 A CN107058104 A CN 107058104A
- Authority
- CN
- China
- Prior art keywords
- oil degradation
- degradation bacteria
- oil
- culture medium
- test tube
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to a kind of cultural method of oil degradation bacteria, it is characterised in that comprises the following steps：(1) oil degradation bacterium culture medium is prepared；(2) by oil degradation bacteria activation in oil degradation bacterium culture medium；(3) the oil degradation bacteria solution diluted is taken to distinguish 200 microlitres etc. with liquid-transfering gun.It is an advantage of the invention that：The oil degradation bacteria cultivated by the present invention, low temperature resistivity can be good, and salt resistant character is good, is suitable for the actual conditions of disposing polluted water in oil, and does not pollute the environment.
Description
Technical field
The present invention relates to a kind of cultural method of oil degradation bacteria.
Background technology
The whole world there are about 1 × 109 ton of oil and products thereof by all means into underground water, surface water every year according to estimates
And in soil, with economic rapid growth, the environmental improvement task of China's oil is also increasingly urgent to add highly effective petroleum degraded
Bacterium is to strengthen the degraded of petroleum hydrocarbons, to reach that the purpose for administering oil polluted environment is imperative.Generally, change
The processing method cost of physics is higher and there is secondary pollution, but microbiological treatment is of relatively low cost, environmental protection.
In field of oilfield sewage treatment, microbial technique is widely used, but the greatest problem existed is the efficient drop that crude oil is substrate
Solve the shortage of oil function bacterium.
The content of the invention
To overcome the defect of prior art, the present invention provides a kind of cultural method of oil degradation bacteria, technology of the invention
Scheme is：
A kind of cultural method of oil degradation bacteria, comprises the following steps：
(1) oil degradation bacterium culture medium is prepared, specific preparation method is as follows：10 parts of peptone, 5 parts of beef extract powder, agar
18 parts of powder, 5 parts of sodium chloride prepares oil degradation bacterium culture medium according to component ratio, and in microwave stove heat, heat time：
2min, heating-up temperature：80 DEG C, untill agar melts completely, it is put into high-pressure sterilizing pot and is sterilized, 121 DEG C of sterilising temp, when
Between 30min, then poured into Biohazard Safety Equipment in sterilized desiccation culture ware, each culture dish pours into 15ml, will be good
The culture dish of culture medium is placed in safety cabinet to cooling, cool time：5h, chilling temperature：25 DEG C of room temperature, can be put into ice after cooling
Case refrigeration is standby；
(2) by oil degradation bacteria activation in oil degradation bacterium culture medium, then to oil degradation in superclean bench
Bacterium carries out flat board coating, while oil degradation bacteria solution concentration is carried out into gradient dilution, 10-2 powers, 10 are diluted to respectively
- 4 powers ,-the 6 of 10 powers and-the 8 of 10 powers.
(3) take the oil degradation bacteria solution diluted to distinguish 200 microlitres with liquid-transfering gun, be injected separately into the stone in culture dish
It is with spreading rod that oil degradation bacteria solution coating is uniform on oil degraded bacterium culture medium, and entered with marking pen in the bottom of culture dish
Rower is noted, and in the constant incubator for being then placed in 35 DEG C, is cultivated, after 1-2 days, observes the growing state of oil degradation bacteria,
The oil degradation bacteria of the different shape grown on culture medium, and reference number are marked respectively with marking pen, then to the stone of mark
Oily degradation bacteria carries out plate streaking；
(4) flat board pulled is put into constant incubator, 35 DEG C, cultivated 1-2 days, the life of oil degradation bacteria on observation flat board
It is long, and dyeing microscopic examination is carried out to the oil degradation bacteria grown, its morphological feature is recorded, the operation is repeated, until under microscope
Untill the oil degradation bacteria form Economical Purification of observation, the oil degradation bacteria of purifying is carried out to carry out in test tube slant culture medium
Inclined-plane line preservation.
Specific dilution process is in described step (2)：10ml distilled water is taken to be put into test tube with syringe, by distilled water
Sterilized, in Biohazard Safety Equipment, 100 microlitres of strain solution taken with liquid-transfering gun, is injected into the test tube of sterile distilled water,
Now, the strain solution concentration in the test tube is 10-2 powers, and strain solution is diluted to by same method respectively 10 for-4 times
Side, 10-6 powers and-the 8 of 10 powers.
Described step (3) plate streaking method is as follows：In Biohazard Safety Equipment, alcolhol burner is lighted, with the top of oese
The oil degradation bacteria of mark sequence number is touched, is drawn on oil degradation bacterium culture medium, the oil degradation bacteria of each sequence number is on culture medium
4th area are drawn, oese is placed on alcolhol burner per standardized area and burnt, is repeated.
The preparation method of described step (4) test tube slant culture medium：Weigh peptone 10g/L, beef extract powder 5g/L, fine jade
Cosmetics 18g/L, sodium chloride 5g/L, are mixed according to proportioning, are heated after mixing in micro-wave oven, the heat time：1-2min, plus
Hot temperature：70-80 DEG C, untill agar powder melts completely, taken with 10ml syringe in 5ml injecting tubes, test tube is put into
2L beaker is wrapped up, and is then placed in high-pressure sterilizing pot and is sterilized, and is sterilized 121 DEG C, time 30min, after sterilizing, by test tube
Pendulum inclined-plane is taken out, the standard of putting on inclined-plane is the 2/3 of of length no more than whole test tube length of nutrient agar on inclined-plane,
Stand 8h, it is to be condensed after, be put into refrigerator cold-storage.
It is an advantage of the invention that：The oil degradation bacteria cultivated by the present invention, low temperature resistivity can be good, and salt resistant character is good, fits
Together in the actual conditions of disposing polluted water in oil, and do not pollute the environment.
Embodiment
The invention will now be further described with reference to specific embodiments, advantages of the present invention and feature will be with description and
It is apparent.But these embodiments are only exemplary, do not constitute any limitation to the scope of the present invention.People in the art
Member to the details and form of technical solution of the present invention it should be understood that can enter without departing from the spirit and scope of the invention
Row modifications or substitutions, but these are changed and replacement is each fallen within protection scope of the present invention.
The present invention relates to a kind of cultural method of oil degradation bacteria, comprise the following steps：
(1) oil degradation bacterium culture medium is prepared, specific preparation method is as follows：Peptone 10g/L, beef extract powder 5g/L, fine jade
Cosmetics 18g/L, sodium chloride 5g/L, oil degradation bacterium culture medium is prepared according to component ratio, and in microwave stove heat, during heating
Between：2min, heating-up temperature：80 DEG C, untill agar melts completely, it is put into high-pressure sterilizing pot and is sterilized, sterilising temp 121
DEG C, then time 30min pours into sterilized desiccation culture ware, each culture dish pours into 15ml in Biohazard Safety Equipment, will
The culture dish of good culture medium is placed in safety cabinet to cooling, cool time：5h, chilling temperature：25 DEG C of room temperature, can put after cooling
Enter refrigerator cold-storage standby；
(2) by oil degradation bacteria activation in oil degradation bacterium culture medium, then to oil degradation in superclean bench
Bacterium carries out flat board coating, while oil degradation bacteria solution concentration is carried out into gradient dilution, 10-2 powers, 10 are diluted to respectively
- 4 powers ,-the 6 of 10 powers and-the 8 of 10 powers.
(3) take the oil degradation bacteria solution diluted to distinguish 200 microlitres with liquid-transfering gun, be injected separately into the stone in culture dish
It is with spreading rod that oil degradation bacteria solution coating is uniform on oil degraded bacterium culture medium, and entered with marking pen in the bottom of culture dish
Rower is noted, and in the constant incubator for being then placed in 35 DEG C, is cultivated, after 1-2 days, observes the growing state of oil degradation bacteria,
The oil degradation bacteria of the different shape grown on culture medium, and reference number are marked respectively with marking pen, then to the stone of mark
Oily degradation bacteria carries out plate streaking；
(4) flat board pulled is put into constant incubator, 35 DEG C, cultivated 1-2 days, the life of oil degradation bacteria on observation flat board
It is long, and dyeing microscopic examination is carried out to the oil degradation bacteria grown, its morphological feature is recorded, the operation is repeated, until under microscope
Untill the oil degradation bacteria form Economical Purification of observation, the oil degradation bacteria of purifying is carried out to carry out in test tube slant culture medium
Inclined-plane line preservation.
Specific dilution process is in described step (2)：10ml distilled water is taken to be put into test tube with syringe, by distilled water
Sterilized, in Biohazard Safety Equipment, 100 microlitres of strain solution taken with liquid-transfering gun, is injected into the test tube of sterile distilled water,
Now, the strain solution concentration in the test tube is 10-2 powers, and strain solution is diluted to by same method respectively 10 for-4 times
Side, 10-6 powers and-the 8 of 10 powers.
Described step (3) plate streaking method is as follows：In Biohazard Safety Equipment, alcolhol burner is lighted, with the top of oese
The oil degradation bacteria of mark sequence number is touched, is drawn on oil degradation bacterium culture medium, the oil degradation bacteria of each sequence number is on culture medium
4th area are drawn, oese is placed on alcolhol burner per standardized area and burnt, is repeated.
The preparation method of described step (4) test tube slant culture medium：Weigh peptone 10g/L, beef extract powder 5g/L, fine jade
Cosmetics 18g/L, sodium chloride 5g/L, are mixed according to proportioning, are heated after mixing in micro-wave oven, the heat time：1-2min, plus
Hot temperature：70-80 DEG C, untill agar powder melts completely, taken with 10ml syringe in 5ml injecting tubes, test tube is put into
2L beaker is wrapped up, and is then placed in high-pressure sterilizing pot and is sterilized, and is sterilized 121 DEG C, time 30min, after sterilizing, by test tube
Pendulum inclined-plane is taken out, the standard of putting on inclined-plane is the 2/3 of of length no more than whole test tube length of nutrient agar on inclined-plane,
Stand 8h, it is to be condensed after, be put into refrigerator cold-storage.
Embodiment 1：
It is simulation oil-polluted water that 6500mg/L, pH are 6.0, oil content 200mg/L in four to take 100mL salinities respectively
In 150mL conical flasks, numbering A, B, C, D access the bacterium solution of the oil-removing bacterias of 0.2mL after purification, D into tri- conical flasks of A, B, C
Do blank control, 25,24h is cultivated respectively, on the basis of D blank, being determined with ultraviolet specrophotometer has content, calculate oil-containing drop
Solution rate, as a result such as following table：
| Numbering | Cultivation temperature/(DEG C) | Containing oil degradation rate/(%) |
| A | 15 | 81.2 |
| B | 25 | 89.5 |
| C | 35 | 86.8 |
Embodiment 2：
100mL salinities are taken to contain greasy dirt for the simulation that 6500mg/L, oil content 200mg/L, pH are configured according to following table respectively
Water is in five 150mL conical flasks, numbering A, B, C, D, E, and 0.2mL removing after purification is accessed into tri- conical flasks of A, B, C, D
The bacterium solution of oily bacterium, E is done under the conditions of blank control, 25 DEG C, and 24h is cultivated respectively, on the basis of E blank, uses ultraviolet specrophotometer
Measure has content, calculates and contains oil degradation rate, as a result such as following table：
| Numbering | pH | Containing oil degradation rate/(%) |
| A | 5.0 | 77.6 |
| B | 6.0 | 81.8 |
| C | 7.0 | 82.9 |
| D | 8.0 | 79.5 |
Embodiment 3：
The plan oil-polluted water that 100mL oil content 200mg/L, pH7.0, salinity according to the form below are set is taken respectively in four
In 150mL conical flasks, numbering A, B, C, D access the bacterium solution of 0.2mL efficient degreasing bacterium into tri- conical flasks of A, B, C, and D does sky
It is white to compare, under the conditions of 25 DEG C, 24h is cultivated respectively, and on the basis of D blank, being determined with ultraviolet specrophotometer has content, and calculating contains
Oil degradation rate, as a result such as following table：
| Numbering | Salinity/(mg/L) | Containing oil degradation rate/(%) |
| A | 6000 | 88.6 |
| B | 7000 | 82.9 |
| C | 8000 | 80.8 |
Embodiment 4：
Take respectively 100mL salinities for 6500mg/L, the plan oil-polluted water of oil content 200mg/L, pH7.0 in four
In 150mL conical flasks, numbering A, B, C, D, into tri- conical flasks of A, B, C, according to the form below connects the oil-removing bacteria of bacterium amount access after purification
Bacterium solution, D does blank control, and the temperature shown according to the form below cultivates 24h respectively, on the basis of D blank, uses uv-spectrophotometric
Meter measure has content, calculates and contains oil degradation rate, as a result such as following table：
| Numbering | Meet bacterium amount/(mL) | Containing oil degradation rate/(%) |
| A | 1 | 80.7 |
| B | 3 | 89.2 |
| C | 5 | 81.9 |
Claims (4)
1. a kind of cultural method of oil degradation bacteria, it is characterised in that comprise the following steps：
(1) oil degradation bacterium culture medium is prepared, specific preparation method is as follows：10 parts of peptone, 5 parts of beef extract powder, agar powder 18
Part, 5 parts of sodium chloride prepares oil degradation bacterium culture medium according to component ratio, and in microwave stove heat, heat time：2min, plus
Hot temperature：80 DEG C, untill agar melts completely, it is put into high-pressure sterilizing pot and is sterilized, 121 DEG C of sterilising temp, time
30min, is then poured into sterilized desiccation culture ware, each culture dish pours into 15ml in Biohazard Safety Equipment, will be trained well
The culture dish for supporting base is placed in safety cabinet to cooling, cool time：5h, chilling temperature：25 DEG C of room temperature, can be put into refrigerator after cooling
Refrigeration is standby；
(2) then oil degradation bacteria activation is entered in oil degradation bacterium culture medium in superclean bench to oil degradation bacteria
Row flat board is coated with, while oil degradation bacteria solution concentration is carried out into gradient dilution, 10-2 powers ,-4 times of 10 are diluted to respectively
Side, 10-6 powers and-the 8 of 10 powers.
(3) take the oil degradation bacteria solution diluted to distinguish 200 microlitres with liquid-transfering gun, be injected separately into the oil drop in culture dish
Solve on bacterium culture medium, it is with spreading rod that oil degradation bacteria solution coating is uniform, and enter rower in the bottom of culture dish with marking pen
In note, the constant incubator for being then placed in 35 DEG C, cultivated, after 1-2 days, the growing state of oil degradation bacteria is observed, with note
Number pen marks the oil degradation bacteria of the different shape grown on culture medium, and reference number respectively, and then the oil of mark is dropped
Solve bacterium and carry out plate streaking；
(4) flat board pulled is put into constant incubator, 35 DEG C, cultivated 1-2 days, the growth of oil degradation bacteria on observation flat board,
And dyeing microscopic examination is carried out to the oil degradation bacteria grown, its morphological feature is recorded, the operation is repeated, until micro- Microscopic observation
Oil degradation bacteria form Economical Purification untill, the oil degradation bacteria of purifying is carried out to carry out inclined-plane in test tube slant culture medium
Line preservation.
2. the cultural method of oil degradation bacteria according to claim 1, it is characterised in that specific in described step (2)
Dilution process is：Take 10ml distilled water to be put into test tube with syringe, distilled water is sterilized, in Biohazard Safety Equipment, use
Liquid-transfering gun takes 100 microlitres of strain solution, is injected into the test tube of sterile distilled water, now, and the strain solution in the test tube is dense
Spend-2 powers for 10, same method, strain solution is diluted to respectively 10-4 powers ,-the 6 of 10 powers and-the 8 of 10 times
Side.
3. the cultural method of oil degradation bacteria according to claim 1, it is characterised in that described step (3) flat board is drawn
Line method is as follows：In Biohazard Safety Equipment, alcolhol burner is lighted, with the oil degradation bacteria that mark sequence number is touched at the top of oese, is drawn
On oil degradation bacterium culture medium, oese is placed on by the oil degradation bacteria of each sequence number in the areas of culture medium Shang Hua tetra- per standardized area
Burn, repeat on alcolhol burner.
4. the cultural method of oil degradation bacteria according to claim 1, it is characterised in that described step (4) test tube is oblique
The preparation method of face culture medium：Weigh peptone 10g/L, beef extract powder 5g/L, agar powder 18g/L, sodium chloride 5g/L, according to
Than being mixed, heated after mixing in micro-wave oven, the heat time：1-2min, heating-up temperature：It is 70-80 DEG C, complete to agar powder
Untill thawing, taken with 10ml syringe in 5ml injecting tubes, the beaker that test tube is put into 2L is wrapped up, and is then placed in height
Pressure autoclave is sterilized, and is sterilized 121 DEG C, time 30min, after sterilizing, and test tube is taken out into pendulum inclined-plane, and the standard of putting on inclined-plane is
The 2/3 of of length no more than whole test tube length of nutrient agar on inclined-plane, stand 8h, it is to be condensed after, be put into refrigerator cold
Tibetan.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710248046.9A CN107058104A (en) | 2017-04-17 | 2017-04-17 | The cultural method of oil degradation bacteria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710248046.9A CN107058104A (en) | 2017-04-17 | 2017-04-17 | The cultural method of oil degradation bacteria |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107058104A true CN107058104A (en) | 2017-08-18 |
Family
ID=59600180
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710248046.9A Pending CN107058104A (en) | 2017-04-17 | 2017-04-17 | The cultural method of oil degradation bacteria |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107058104A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104388312A (en) * | 2014-12-10 | 2015-03-04 | 青岛农业大学 | Screening method for petroleum degrading bacteria, method for preparing petroleum degrading bacteria inoculant from screened bacteria, and application of inoculant |
| CN104830708A (en) * | 2015-02-02 | 2015-08-12 | 天津科技大学 | Crude oil degrading bacteria strain and application thereof |
-
2017
- 2017-04-17 CN CN201710248046.9A patent/CN107058104A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104388312A (en) * | 2014-12-10 | 2015-03-04 | 青岛农业大学 | Screening method for petroleum degrading bacteria, method for preparing petroleum degrading bacteria inoculant from screened bacteria, and application of inoculant |
| CN104830708A (en) * | 2015-02-02 | 2015-08-12 | 天津科技大学 | Crude oil degrading bacteria strain and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| XING-BIAOWANG ET AL.: "Degradation of petroleum hydrocarbons (C6-C40) and crude oil by a novel Dietzia strain", 《BIORESOURCE TECHNOLOGY》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Obana et al. | Effect of Nostoc sp. on soil characteristics, plant growth and nutrient uptake | |
| CN106755286B (en) | A method of testing oil extraction waste water bio-toxicity using Vibrio-qinghaiensis sp. Q67 | |
| CN104498404B (en) | Bacillus CP-1 for lead-chromium pollution restoration and application of bacillus CP-1 | |
| CN103266073B (en) | Sedum alfredii endophyte and application thereof | |
| AU2019460845B2 (en) | High-efficiency petroleum degrading bacterium TDYN1T and use thereof | |
| CN103205378B (en) | Oil-degradation oil-and-salt tolerance bacterial strain, and screening method and application thereof | |
| CN101948787A (en) | Method for selecting rhamnolipid-producing bacterium and used medium | |
| CN104001712A (en) | Method for repairing petroleum-contaminated soil through combination of alkali grass and microbes | |
| CN107058104A (en) | The cultural method of oil degradation bacteria | |
| CN113046267B (en) | Rhodococcus ruber and application thereof | |
| CN109251880A (en) | A kind of Bacillus cereus and its application in improvement water systems'phosphorus pollution | |
| CN105875406B (en) | A kind of induction of Borneolum tree callus and enrichment procedure | |
| CN107058105A (en) | The cultural method of nitrite degradation bacterium | |
| CN106544301A (en) | The bacillus licheniformis of one plant of high temperature resistant degrading crude oil galactopoiesis agent and its application | |
| CN103695332B (en) | A kind of contaminated by diesel oil type place microbiological deterioration bacterium | |
| CN110283759A (en) | The separation and application of one plant of grease degrading strain Kosakonia cowaniiIUMR B67 | |
| CN106957800A (en) | The cultural method of COD degradation bacterium | |
| CN106978344A (en) | The cultural method of Phenol-degrading Bacteria Strains | |
| CN106978345A (en) | The cultural method of biological demulsifying agent producing strains | |
| CN110317745A (en) | Ralstonia pickettii M1 bacterial strain and its application in degradation phenanthrene and biphenyl | |
| CN106939285A (en) | The cultural method of deodorization bacterium | |
| CN105154353A (en) | Bacillus subtilis and application thereof in greenhouse soil remediation | |
| CN106947695A (en) | The cultural method of ammonia nitrogen degradation bacterium | |
| CN104403968A (en) | Bacillius firmus GY-49, screening method and applications thereof | |
| CN104046580A (en) | Sphingobacterium strain for degrading polycyclic aromatic hydrocarbon organic pollutant and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170818 |
|
| RJ01 | Rejection of invention patent application after publication |