CN107629976A - A kind of microorganism and its microbial inoculum and application - Google Patents
A kind of microorganism and its microbial inoculum and application Download PDFInfo
- Publication number
- CN107629976A CN107629976A CN201610565369.6A CN201610565369A CN107629976A CN 107629976 A CN107629976 A CN 107629976A CN 201610565369 A CN201610565369 A CN 201610565369A CN 107629976 A CN107629976 A CN 107629976A
- Authority
- CN
- China
- Prior art keywords
- huo shi
- oil
- enterobacterias
- tween
- microbial
- 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
Abstract
The invention provides a kind of microorganism and its microbial inoculum and application.The microorganism is Huo Shi enterobacterias Y 29, and bacterial strain deposit number is CCTCC NO:M2016049.The preparation method of the microbial inoculums of Huo Shi enterobacterias Y 29 of the present invention is：First the Huo Shi enterobacterias Y 29 of preservation is activated, cultivates seed liquor and then fermented, centrifuge and obtain thalline, thalline and tween solution are mixed to get the microbial inoculums of Huo Shi enterobacterias Y 29 according to every 60mL tween solutions (0.25%) containing 1g thalline.The Huo Shi enterobacterias Y 29 and its microbial inoculum of the present invention has obvious biological prosthetic effect to oil pollution, has preferable degradation effect to the oil in crude oil and oil-polluted soils.Except this, the microbial inoculum also has with short production cycle, and cost is cheap, using simple, non-secondary pollution, the advantages that effect on environment is small, enriches existing oil pollution degradation bacteria strains resource, has boundless application prospect in soil petroleum pollution reparation.
Description
Technical field
The invention belongs to technical field of microbiology, is related to a kind of microorganism and its microbial inoculum and application.
Background technology
With being continuously increased for human society oil product demand, oil and its product enter environment with number of ways,
Such as oil plant oily waste water, external oil plant often process 1 ton of crude oil, produce 0.5-1 ton oily waste waters, and China's oil plant by
More in refining mink cell focus, refining process is complicated, often processes 1 ton of crude oil, produces 0.7-3.5 ton oily waste waters.According to statistics, China is refined
Oily production capacity has reached annual 2.4 × 108Ton, actual processing amount are annual 1.6 × 108Ton or so, by this calculating, often produce per year
Go out oily waste water and be up to 1.12-5.6 × 108Ton.These oily waste waters cause serious pollution to soil and water quality, threaten people
The health of class；For another example, ocean water body oily pollution is a kind of current global serious marine pollution, be concentrated mainly on river mouth,
Around bay offshore waters, offshore oilfield；And for example, during oil exploration and exploitation, accumulating, refining etc., due to operating not
When or the reason such as accident leakage and maintenance, can also make it that some oil are unrestrained out, cause the pollution of soil, oilfield development process
In gas blowout accident, petroleum pipeline and oil storage tank leakage accident, oil field equipment maintenance etc. can all cause oil pollution.Therefore,
The research of oil pollution recovery technique is increasingly subject to pay attention to and paid close attention to.
The method of existing processing oil pollution mainly has peripheral doses, chemical remediation and biological prosthetic, and peripheral doses include
(1) fence method：Oil is prevented to spread across the sea；(2) oil skimmer：On the basis of nature of petroleum is not changed, oil is carried out
Recovery；(3) oil absorption material：With oil loving material, adsorption recovery is carried out to oil.Chemical remediation includes：(1) dispersant：Can
Effectively to reduce the surface tension between oil and seawater, so as to which oil be disperseed；(2) fuel thickener：Oil can be condensed into sticky
Shape or g., jelly-like, so as to prevent oil from spreading；(3) other chemicals.Biological renovation method is nineteen ninety-five earliest by Glazer
Itd is proposed with Nikaido, pass through the process of harmful waste in microbial degradation or removing environment, the pollution of catalytic degradation environmental toxic
Thing, reduce or finally eliminate the controlled or spontaneous process of environmental pollution.Hydrocarbon degradation bacterium just has found early in early 20th century, according to report
Road can have 79 bacterium category, 9 blue-green algae category, 103 fungis and 14 algaes by the use of hydro carbons as sole carbon source and the energy
Category.
Compare three kinds of restorative procedures, peripheral doses also destroy the component and knot of soil while soil pollutant is destroyed
Structure, and it is costly.Chemical remediation effect is preferable, but used chemical agent can produce secondary pollution, and limiting it should
Use scope.Bioremediation technology is considered as a kind of green, non-secondary pollution, efficient, low toxicity, low-residual, can thoroughly dropped
Solve the oil pollution restorative procedure with development prospect of pollutant.Utilize the degradable different kinds of petroleum hydrocarbon class group of functional microorganism
Point.
Microbe remedying oil-polluted soils at home and abroad have been reported that, a patented technology of BGI companies of the U.S.
Exactly promote the degraded of petroleum hydrocarbon using mixed bacterial；Application No. 2015104518635,2015103014570,
2015102180395 patent application document discloses oil degradation microbial inoculum and its production and use, the microorganism being related to
Including Chryseobacterium sp, shank bacterium, achromobacter, Burkholderia, food alkane Gordonia bronchialis NR 39, the friendly and of Gordonia bronchialis NR 70
Acinetobacter calcoaceticus BS6, available for the reparation of oil-polluted soils, there is preferable oil degradation effect.2008, Su Ying
Deng from Shengli Oil Field sewage, enrichment culture is carried out with artificial seawater culture medium and obtains one plant of suitable petroleum pollution in ocean reparation
Bacterial strain HB-1, is accredited as acinetobacter.2010, Zhang Yuemei etc. screened to obtain 50 plants using oil as sole carbon source from the arctic
Thermophilic cold degradation bacteria, wherein 3 plants of degradation rate highest are all Pseudoalteromonas.2015, Zhang Aijun etc. screened one from the Bohai Sea
Strain Pseudoalteromonas.Raghukumar etc. research finds that cyanobacteria can drop under artificial seawater condition of culture in ocean
Solve crude oil.
But it is used for that degraded oil pollutant is unprecedented reports for work for Huo Shi enterobacterias.In addition, oil pollution at present is micro-
The problem of biological prosthetic middle maximum is that biodegradability is not ideal enough, less stable.
The content of the invention
In order to improve in existing petroleum microorganism recovery technique biodegradability deficiency, the problem of stability difference, this hair
Bright purpose is the micro- life for providing a kind of microorganism Huo Shi enterobacteria Y-29 biological prosthetic for oil pollution and its preparation
Thing microbial inoculum；
The present invention also aims to provide the application of mentioned microorganism and its microbial inoculum in oil pollution is biological prosthetic.
The purpose of the present invention is achieved by the following technical programs：
A kind of Huo Shi enterobacterias (Enterobacter hormaechei) Y-29, the Huo Shi enterobacterias Y-29 is in preservation
Unit preservation, depositary institution's title：China typical culture collection center (CCTCC), depositary institution address：Wuhan, China Wuhan
University, postcode：430072；Preservation date：On January 18th, 2016, culture presevation numbering is CCTCC NO:M 2016049；Point
Class is named：Huo Shi enterobacterias Y-29 (Enterobacter hormaechei Y-29).
Above-mentioned Huo Shi enterobacterias Y-29 16S rDNA base sequence such as SEQ ID NO：Shown in 1.
The present invention also provides a kind of microbial bacterial agent, and the microbial bacterial agent contains above-mentioned Huo Shi enterobacterias Y-29, described
Huo Shi enterobacterias Y-29 is the active component of the microbial bacterial agent, can also add auxiliary element, such as tween, talcum powder, silicon
Diatomaceous earth etc..
The present invention also provides a kind of preparation method of Huo Shi enterobacterias Y-29 microbial inoculums, and it comprises the following steps：
Step 1, Huo Shi enterobacterias Y-29 is inoculated in LB solid mediums, 30-36h is cultivated at 35-37 DEG C, obtains
To the bacterial strain of activation；
Step 2, by the inoculation activated in step 1 into LB fluid nutrient mediums, vibrated under 37 DEG C, 140r/min
36h is cultivated, obtains seed liquor；
Step 3, the seed liquor in step 2 is inoculated with according to the 10% of LB fermentation medium volumes, in 37 DEG C,
140r/min shaken cultivations 36-40h, isolated thalline；
Step 4, the thalline in step 3 and tween solution are contained into 1g thalline according to every 60mL tween solutions and are mixed to get
The Huo Shi enterobacteria Y-29 microbial inoculums.
In above-mentioned preparation method, it is preferable that the preparation raw material of the LB solid mediums includes：Contain in per 1000mL water
5g yeast extracts, 10g tryptones, 10g sodium chloride, 20g agar powders；
The preparation raw material of the LB fluid nutrient mediums includes：Contain 5g yeast extracts, 10g tryptoses in per 1000mL water
Peptone, 10g sodium chloride；
The preparation raw material of the LB fermentation mediums includes：Contain 5g yeast extracts, 10g tryptoses in per 1000mL water
Peptone, 10g sodium chloride.
In above-mentioned preparation method, the compound method of the LB solid mediums is：Yeast extract 5g, tryptone 10g,
Sodium chloride 10g, agar powder 20g, 1000mL mixed dissolutions are added water to, and pH value is adjusted to 7.0-7.2 with sodium hydroxide, in 121
Cooled down at DEG C after sterilizing 20min；
The compound method of the LB fluid nutrient mediums is：Yeast extract 5g, tryptone 10g, sodium chloride 10g, add water
PH value is adjusted to 7.0-7.2 to 1000mL mixed dissolutions, and with sodium hydroxide, is cooled down after the 20min that sterilized at 121 DEG C；
The compound method of the LB fermentation mediums is：Yeast extract 5g, tryptone 10g, sodium chloride 10g, add water
PH value is adjusted to 7.0-7.2 to 1000mL mixed dissolutions, and with sodium hydroxide, is cooled down after the 20min that sterilized at 121 DEG C.
In above-mentioned preparation method, it is preferable that tween is Tween 80 in the tween solution, and the tween solution is by every
0.25mL Tween 80s are added in 100mL physiological saline to be prepared.
In the microbial inoculum of above-mentioned preparation, using addition surface active agent tween Tween-80, oil can be evacuated into small oil
Drop, increase the contact area of microorganism and oil, so as to accelerate degraded of the microorganism to oil.
Present invention also offers the Huo Shi enterobacteria Y-29 microbial inoculums that above-mentioned preparation method is prepared, the Huo Shi enterobacterias Y-
29 microbial inoculums are liquid, and bacteria concentration is 1.0 × 108Cfu/mL, optical density OD600=0.88-0.90.
The present invention also provides applications of the above-mentioned Huo Shi enterobacterias Y-29 on oil pollution is biological prosthetic.
The present invention also provides application of the above-mentioned microbial bacterial agent on oil pollution is biological prosthetic.
The present invention also provides application of the above-mentioned Huo Shi enterobacteria Y-29 microbial inoculums on oil pollution is biological prosthetic.
Huo Shi enterobacterias Y-29 provided by the invention is the separation screening out of Jianghan Plain oil-polluted soils, to oil
With preferable degradation, there is good biological prosthetic effect to oil-polluted soils, enrich existing oil pollution drop
Strain resource is solved, important reference is provided for the microorganism remediation of oil pollution.
Bacterial strain Y-29 of the present invention is Gram-negative bacteria, in direct rod shape, both ends blunt circle, peritrichous motion.
For the bacterium colony formed on LB flat boards into circle, edge is irregular, and milky is opaque to slightly yellow.Physio-biochemical characteristics：It is facultative to detest
Oxygen, glucose fermentation, sour aerogenesis is produced, contact enzyme positive.Sequence will have been reported on 16S rDNA sequencing results and Genbank
(No.JN993998.1) it is compared, it is final to determine that bacterial strain Y-29 is Huo Shi enterobacterias (Enterobacter
hormaechei)。
Huo Shi enterobacterias Y-29 microbial inoculums provided by the invention are with short production cycle, only need shaken cultivation 36-40h；Cost is cheap,
It is mixed with using addition surface active agent tween with Huo Shi enterobacteria bacteria suspensions；Using simple, microbial inoculum need to be only put into stone
In oil contaminants；Non-secondary pollution, effect on environment are small.
Beneficial effects of the present invention：
The Huo Shi enterobacterias Y-29 of the present invention has obvious biological prosthetic effect to oil pollution, to the degradation rate of crude oil
47.03% is can reach, the Huo Shi enterobacteria Y-29 microbial inoculums of preparation can reach 52.07% to the degradation rate of crude oil；When soil oil
When pollution rate is 3%-7%, Huo Shi enterobacterias Y-29 is to the petroleum degradation rate in oil-polluted soils up to more than 60%；Remove
This, microbial inoculum prepared by Huo Shi enterobacterias Y-29 of the invention has with short production cycle, and cost is cheap, using simple, without secondary dirt
Dye, the advantages that effect on environment is small, existing oil pollution degradation bacteria strains resource is enriched, is had in soil petroleum pollution reparation
Boundless application prospect.
Brief description of the drawings
Fig. 1 be the embodiment of the present invention 1 in Huo Shi enterobacterias Y-29 LB cultured on solid medium colonial morphology.
Embodiment
In order to which technical characteristic, purpose and the beneficial effect of the present invention is more clearly understood, now to the skill of the present invention
Art scheme carry out it is described further below, but it is not intended that to the present invention can practical range restriction.
The Huo Shi enterobacterias Y-29 of embodiment 1 is screened and identification
1st, crude oil biological repairs the separation of bacterial strain
It is directly separated method：
(1) 10g fresh soil samples are weighed to be added in the triangular flask equipped with 90mL sterilized waters, constant temperature oscillation 30min (30 DEG C,
200r/min)；
(2) add aqua sterilisa and carry out gradient dilution into 10-6、10-7、10-8, the bacterium solutions of three kinds of different dilution factors is taken respectively
0.1mL is coated on oil-containing (0.5%) inorganic salts flat board, is coated with uniformly with sterile spatula, each dilution factor is repeated 3 times, 28 DEG C
Under incubated 4-5d；Minimal medium formula：NaNO32g, K2HPO41g, K2HPO4·3H2O 0.5g, NaCl 0.5g,
MgS04·7H2O 0.1g, CaCl20.01g, FeSO4·7H2O 0.01g, add H2O to 1000mL, PH=7.
(3) fast with the oese picking speed of growth, well-grown bacterium colony is rule on LB solid plates, constant temperature at 28 DEG C
Cultivate 1-2d；The single bacterium colony of different colours and form, line purifying, common picking single bacterium colony 117 are selected after flat board grows bacterium colony
Strain, is transferred and is cultivated on LB flat boards, and 4 DEG C after purifying bacterial strain access test tube slant culture are saved backup.
Enrichment and separation method：
(1) 10g oil pollution soil samples are weighed to be added in the 300mL conical flasks containing 90mL sterilized waters, in 30 DEG C,
Constant-temperature shaking culture 3-4h under the conditions of 150r/min, the bacterium in soil sample is dispersed in water, stands 30min；
(2) this Soil Slurry of 1mL is taken to be inoculated into the LB fluid nutrient mediums equipped with 100mL oil-containings 0.3%, 30 DEG C,
Constant temperature oscillation enrichment culture 5d under the conditions of 150r/min；
(3) draw above-mentioned enrichment culture liquid 10mL to be added in the LB fluid nutrient mediums equipped with 100mL oil-containings 0.5%, 30
DEG C, constant temperature oscillation enrichment culture 5d under the conditions of 150r/min, repeat this step 2 time；
(4) enrichment culture liquid is suitably diluted, then drawing 0.1mL dilutions, coating is equal to LB solid plates
Even, each diluted concentration is repeated 3 times；30 DEG C of incubated 2-4d；
(5) fast with the oese picking speed of growth, well-grown bacterium colony is rule on LB solid plates, 28 DEG C, constant temperature
1-2d is cultivated, the single bacterium colony of different colours and form, line purifying, common picking single bacterium colony 37 are selected after flat board grows bacterium colony
Strain, is transferred and is cultivated on LB flat boards, and 4 DEG C after purifying bacterial strain access test tube slant culture are saved backup.
2nd, crude oil biological repairs the screening of bacterial strain
(1) preparation of bacteria suspension：By isolated bacterial strain, a ring is chosen into LB fluid nutrient mediums with transfer needle, 200r/
M, 28 DEG C, shaking table culture 24h；
(2) domestication culture：Drawing lmL, cultured bacteria suspension is added to 50mL oil-containings (0.0300g-0.0500g) nothing
In machine salt culture medium, shaken cultivation is carried out on 30 DEG C, 200r/min constant-temperature table.To reduce evaluated error, each sample
Do three Duplicate Samples, while using the shaking flask of not microbe inoculation as control to deduct abiotic influence；
(3) measure of sample：By above-mentioned nutrient solution, carefully move into separatory funnel, with (the 60-90 DEG C of boiling of appropriate petroleum ether
Journey) cleaning conical flask 2-3 time, and washing lotion is moved into separatory funnel, then fully vibration separatory funnel 3min, stand and be allowed to point
Layer, lower floor's aqueous is discarded, and upper liquid is collected in 100mL volumetric flasks, with a certain amount of petroleum ether funnel, its cleaning solution
It is collected in same volumetric flask, and graduation mark is diluted to petroleum ether, after shaking up and standard series is together in ultraviolet spectrometry light
The upper progress colorimetric at 225nm wavelength of degree meter, using petroleum ether used in constant volume dilutes as reference, OD values are measured, using standard curve,
Calculate oil content；
(4) petroleum degradation rate is calculated, formula is as follows：
Wherein：M1For the quality of before processing oil
M2For the residual quantity of oil after processing
B1For the quality of blank control before processing oil
B2The residual quantity of oil after being handled for blank control
The drafting of oil standard curve：
Into 7 50mL volumetric flasks, 0,2.00,4.00,8.00,10.00,12.00 and 25.00mL standard oils are separately added into
Using liquid, graticule is diluted to petroleum ether.Given wavelength is being selected, absorbance is determined by reference of petroleum ether, after blank correction,
Draw standard curve.
It is as shown in table 1 according to the method for above-mentioned oil Specification Curve of Increasing, measurement result.
Table 1
| Sequence number | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| Concentration (mg/L) | 0 | 4 | 8 | 16 | 20 | 24 | 50 |
| Light absorption value A | 0 | 0.0739 | 0.1439 | 0.3091 | 0.4209 | 0.4745 | 0.9863 |
Standard curve is drawn according to the result of standard curve determination, its standard curve relationship equation is y=50.199x+
0.1558, R2=0.9986, absorbance is linearly related notable with concentration.
Bacterial strain after purification is screened.Bacterial strain is inoculated into oil-containing (0.03g-0.05g) inorganic salts respectively during screening
In culture medium, 30 DEG C, shaken cultivation 7d is carried out on 200r/min shaking tables, Residual oil OD225 values are measured with ultraviolet specrophotometer, with
The minimal medium of oil-containing after concentration and residual quantity that Residual oil is conversed by standard curve, does not calculate degraded for control
Rate, as a result as shown in table 2, table 2 are the selection result of oil pollution degradation bacteria strains.Above-mentioned minimal medium formula is：NaNO3
2g, K2HPO41g, K2HPO4·3H2O 0.5g, NaCl 0.5g, MgSO4·7H2O0.1g, CaCl20.01g, FeSO4·7H2O
0.01g, add H2O to 1000mL, PH=7.
Table 2
It can be seen that by the experimental result of table 2：After cultivating 7d in inorganic salts oil-containing culture medium, 33 plants of bacterial strains are to crude oil table
Reveal different degrees of degradation, wherein bacterial strain Y-29 degradation rate is up to 47.03%, next to that Y-32 and Y-30, drop
Solution rate is respectively 36.89% and 32.59%.
3rd, Huo Shi enterobacterias Y-29 identification
Physiology and biochemistry identification is carried out to the bacterial strain Y-29 of screening, is defined as Gram-negative bacteria, Enterobacter, colonial morphology
As shown in figure 1, its bacterium colony, into circle, edge is irregular, milky is opaque to slightly yellow.Then total gene of bacterial strain is extracted
Group DNA is its 16S rDNA sequence of template amplification.
(1) primer is designed：
Sense primer：27F：5 '-AGAGTTTGATCCTGGCTCAG-3 ' are (referring to SEQ ID No：Shown in 2)；
Anti-sense primer：1429R：5 '-GGTTACCTTGTTACGACTT-3 ' are (referring to SEQ ID No：Shown in 3)；
(2) pcr amplification reaction system is (15 μ L)：
DNA profiling：0.6μL；H2O：8.57μL；Buffer：1.65μL、dNTP mixture：0.66μL；27F：0.33μ
L；1429R：0.33μL；The μ L of Tsg archaeal dna polymerases 0.11；
(3) condition of the PCR amplifications is：
94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 30s；50 DEG C of degeneration 45s；72 DEG C of extension 100s；Circulation 35 times；72 DEG C of extensions
7min；
(4) after PCR amplifications terminate, simultaneously purified pcr product, sequencing, its 16S rDNA gene order such as SEQ ID are reclaimed
No：Shown in 1, sequencing result is contrasted with the sequence (No.JN993998.1) reported on Genbank, by bacterial strain Y-29
It is accredited as Huo Shi enterobacterias (Enterobacter hormaechei).
The preparation of the Huo Shi enterobacteria Y-29 microbial inoculums of embodiment 2
A kind of preparation method of Huo Shi enterobacterias Y-29 microbial inoculums is present embodiments provided, it comprises the following steps：
Step 1, Huo Shi enterobacterias Y-29 is inoculated in LB solid mediums, 30-36h is cultivated at 35-37 DEG C, obtains
To the bacterial strain of activation；
LB solid medium compound methods are：Yeast extract 5g, tryptone 10g, sodium chloride 10g, agar powder 20g,
Add water mixed dissolution, and pH value is adjusted to 7.0-7.2 with sodium hydroxide, sterilize 20min at 121 DEG C.
Step 2, by the inoculation activated in step 1 into LB fluid nutrient mediums, vibrated under 37 DEG C, 140r/min
36h is cultivated, obtains seed liquor；
LB fluid nutrient medium compound methods are：Yeast extract 5g, tryptone 10g, sodium chloride 10g, add water mixing molten
Solution, and pH value is adjusted to 7.0-7.2 with sodium hydroxide, sterilize 20min at 121 DEG C.
Step 3, the seed liquor in step 2 is inoculated with according to the 10% of LB fermentation medium volumes, in 37 DEG C,
140r/min shaken cultivation 36-40h, obtain bacteria suspension, and bacteria suspension is centrifugally separating to obtain into thalline；
Fermentation medium compound method is：Yeast extract 5g, tryptone 10g, sodium chloride 10g, add water mixed dissolution,
And pH value is adjusted to 7.0-7.2 with sodium hydroxide, sterilize 20min at 121 DEG C.
Step 4, by the thalline in step 3 and tween solution according to Tween 80-physiology that every 60mL concentration is 0.25%
In the solution of salt solution the Huo Shi enterobacteria Y-29 microbial inoculums are mixed to get containing 1g thalline.The bacterium of the Huo Shi enterobacteria Y-29 microbial inoculums is dense
Spend for 1.0 × 108Cfu/mL, optical density OD600=0.88-0.90.
Y-29 microbial inoculums are used for oil degradation, with embodiment 1, Y-29 drops method after adding Tween 80 (0.25%) to oil
Solution rate is 52.07%.
Degradeds of the Huo Shi enterobacterias Y-29 of embodiment 3 to oil-polluted soils
By the sterile soil of 3.0%, 5.0%, 7.0%, 9.0%, 11.0% different oil pollutions respectively weigh respectively 10g in
In triangular flask, it is inoculated with respectively and has cultivated 24h bacterium solution 4mL and cultivate 45d under normal temperature, using the soil without oil pollution as control.
Levigate after soil sample is dried at 105 DEG C to cross 0.25mm sieves, each sample that handles is weighed the native 5g of drying, carried using soxhlet extraction
After taking, 250mL is settled under 225nm wavelength, is measured with ultraviolet spectrophotometry, if sample petroleum concentration exceeds standard
Curve ranges determine after then carrying out suitably dilution.3.0% each processing takes 1.5mL to be settled to 50mL；5.0% each processing takes 1mL to determine
Hold to 50mL and dilute 50 times；7.0%th, 9.0%, 11.0% each processing takes 0.5mL to be settled to 50mL to dilute 100 times.Utilize
The content of soil petrochina after degraded culture is calculated in mark song.
Huo Shi enterobacterias Y-29 is as shown in table 3 to the degradation experiment result of oil-polluted soils, it may be seen that bacterial strain Y-
The soil of 29 pairs of oil pollutions has higher degradation rate, when soil petroleum pollution rate is 3.0%, 5.0%, 7.0%, oil
Degradation rate is all higher than 60%.
Table 3
In summary, Huo Shi enterobacterias Y-29 of the invention has obvious biological prosthetic effect to oil pollution, to original
The degradation rate of oil can reach 47.03%；The Huo Shi enterobacteria Y-29 microbial inoculums of preparation can reach 52.07% to the degradation rate of crude oil；
When soil petroleum pollution rate is 3%-7%, Huo Shi enterobacterias Y-29 is reachable to the petroleum degradation rate in oil-polluted soils
More than 60%；Except this, the microbial inoculum also have it is with short production cycle, cost is cheap, uses simple, non-secondary pollution, effect on environment
The advantages that small, existing oil pollution degradation bacteria strains resource is enriched, there is boundless answer in soil petroleum pollution reparation
Use prospect.
Claims (10)
- A kind of 1. Huo Shi enterobacterias Y-29, it is characterised in that：The culture presevation numbering of the Huo Shi enterobacterias Y-29 is CCTCC NO: M2016049。
- 2. Huo Shi enterobacterias Y-29 according to claim 1, it is characterised in that：The 16S of the Huo Shi enterobacterias Y-29 RDNA base sequence such as SEQ ID NO：Shown in 1.
- A kind of 3. microbial bacterial agent, it is characterised in that：The microbial bacterial agent contains the Huo Shi enterobacterias described in claim 1 or 2 Y-29。
- 4. a kind of preparation method of Huo Shi enterobacterias Y-29 microbial inoculums, it comprises the following steps：Step 1, Huo Shi enterobacterias Y-29 is inoculated in LB solid mediums, 30-36h is cultivated at 35-37 DEG C, is lived The bacterial strain of change；Step 2, by the inoculation activated in step 1 into LB fluid nutrient mediums, the shaken cultivation under 37 DEG C, 140r/min 36h, obtain seed liquor；Step 3, the seed liquor in step 2 is inoculated with according to the 10% of LB fermentation medium volumes, in 37 DEG C, 140r/ Min shaken cultivations 36-40h, isolated thalline；Step 4, the thalline in step 3 and tween solution are contained into 1g thalline according to every 60mL tween solutions and are mixed to get this suddenly Family name's enterobacteria Y-29 microbial inoculums.
- 5. preparation method according to claim 4, it is characterised in thatThe preparation raw material of the LB solid mediums includes：In per 1000mL water containing 5g yeast extracts, 10g tryptones, 10g sodium chloride, 20g agar powders；The preparation raw material of the LB fluid nutrient mediums includes：In per 1000mL water containing 5g yeast extracts, 10g tryptones, 10g sodium chloride；The preparation raw material of the LB fermentation mediums includes：In per 1000mL water containing 5g yeast extracts, 10g tryptones, 10g sodium chloride.
- 6. preparation method according to claim 4, it is characterised in that：Tween is Tween 80 in the tween solution, and this is told Warm solution is prepared by adding 0.25mL Tween 80s in every 100mL physiological saline.
- 7. the Huo Shi enterobacteria Y-29 microbial inoculums that preparation method described in claim 4-6 is prepared, it is characterised in that：The Huo Shi intestines The bacteria concentration of bacillus Y-29 microbial inoculums is 1.0 × 108Cfu/mL, optical density OD600=0.88-0.90.
- 8. applications of the Huo Shi enterobacterias Y-29 on oil pollution is biological prosthetic described in claim 1 or 2.
- 9. application of the microbial bacterial agent on oil pollution is biological prosthetic described in claim 3.
- 10. application of the Huo Shi enterobacteria Y-29 microbial inoculums on oil pollution is biological prosthetic described in claim 7.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610565369.6A CN107629976A (en) | 2016-07-18 | 2016-07-18 | A kind of microorganism and its microbial inoculum and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610565369.6A CN107629976A (en) | 2016-07-18 | 2016-07-18 | A kind of microorganism and its microbial inoculum and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107629976A true CN107629976A (en) | 2018-01-26 |
Family
ID=61113091
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610565369.6A Pending CN107629976A (en) | 2016-07-18 | 2016-07-18 | A kind of microorganism and its microbial inoculum and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107629976A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108441450A (en) * | 2018-04-10 | 2018-08-24 | 暨南大学 | It is a kind of to have both Soluble phosphorus and the composite bacteria agent and its application that DEHP is acted on of degrading |
| CN109370950A (en) * | 2018-11-29 | 2019-02-22 | 重庆工商大学 | A kind of light petroleum alkane degradation enterobacteria and its preparation method and application |
| CN113186131A (en) * | 2021-04-30 | 2021-07-30 | 广州绿曦生物科技有限公司 | Alga-lysing microbial agent and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070249034A1 (en) * | 2003-11-13 | 2007-10-25 | Brigmon Robin L | Surfactant biocatalyst for remediation of recalcitrant organics and heavy metals |
| CN101948763A (en) * | 2009-10-13 | 2011-01-19 | 中国农业科学院饲料研究所 | High-efficiency compound microbial inoculum for fermenting beds, and preparation method and application thereof |
| CN104649848A (en) * | 2015-03-06 | 2015-05-27 | 天津理工大学 | Solid bacterial fertilizer for remedying petroleum polluted saline alkali soil and preparation method of solid bacterial fertilizer |
| CN104726370A (en) * | 2015-02-16 | 2015-06-24 | 湖南农业大学 | Enterobacter with crude oil degradation effect and application of enteric bacilli |
| CN104928207A (en) * | 2015-04-30 | 2015-09-23 | 太原理工大学 | Enterobacter hormaechei CL2013 as well as method for preparing hexavalent chromium restoring bactericide |
-
2016
- 2016-07-18 CN CN201610565369.6A patent/CN107629976A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070249034A1 (en) * | 2003-11-13 | 2007-10-25 | Brigmon Robin L | Surfactant biocatalyst for remediation of recalcitrant organics and heavy metals |
| CN101948763A (en) * | 2009-10-13 | 2011-01-19 | 中国农业科学院饲料研究所 | High-efficiency compound microbial inoculum for fermenting beds, and preparation method and application thereof |
| CN104726370A (en) * | 2015-02-16 | 2015-06-24 | 湖南农业大学 | Enterobacter with crude oil degradation effect and application of enteric bacilli |
| CN104649848A (en) * | 2015-03-06 | 2015-05-27 | 天津理工大学 | Solid bacterial fertilizer for remedying petroleum polluted saline alkali soil and preparation method of solid bacterial fertilizer |
| CN104928207A (en) * | 2015-04-30 | 2015-09-23 | 太原理工大学 | Enterobacter hormaechei CL2013 as well as method for preparing hexavalent chromium restoring bactericide |
Non-Patent Citations (3)
| Title |
|---|
| 李鱼: "《自然水体生物膜上铁、锰氧化物的形态及其吸附机理研究》", 31 December 2008 * |
| 蒋磊等: "生物表面活性剂及其在生物降解中的应用", 《湖北农业科学》 * |
| 赵乃昕等: "《医学细菌名称及分类鉴定》", 31 December 2013 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108441450A (en) * | 2018-04-10 | 2018-08-24 | 暨南大学 | It is a kind of to have both Soluble phosphorus and the composite bacteria agent and its application that DEHP is acted on of degrading |
| CN108441450B (en) * | 2018-04-10 | 2020-05-08 | 暨南大学 | Complex microbial inoculant with phosphorus dissolving and DEHP degrading functions and application thereof |
| CN109370950A (en) * | 2018-11-29 | 2019-02-22 | 重庆工商大学 | A kind of light petroleum alkane degradation enterobacteria and its preparation method and application |
| CN113186131A (en) * | 2021-04-30 | 2021-07-30 | 广州绿曦生物科技有限公司 | Alga-lysing microbial agent and application thereof |
| CN113186131B (en) * | 2021-04-30 | 2023-10-27 | 广州绿曦生物科技有限公司 | Algicidal microbial agent and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103436464B (en) | Low temperature-resistant petroleum-degrading bacillus sp. strain, culture method and application thereof | |
| CN104745511B (en) | Composite bacteria agent for repairing heavy oil pollution coastline and preparation method and application | |
| CN102250798B (en) | Pseudomonas and microbial agent as well as applications thereof | |
| CN106493167A (en) | Bacillus licheniformis and microbial inoculum and their application and heavy metal-passivated method | |
| CN102994428B (en) | One strain ocean surfactant producing bacteria strain LHOD-1 and application thereof | |
| CN102277312A (en) | Strain for degrading polycyclic aromatic hydrocarbons at low temperature and application in bioremediation of groundwater in petroleum hydrocarbon polluted places thereof | |
| CN103205378B (en) | Oil-degradation oil-and-salt tolerance bacterial strain, and screening method and application thereof | |
| CN106497810B (en) | A kind of method of germ oligotrophy unit cell, the microbial inoculum containing the bacterium and its application and diesel oil of degrading | |
| CN110846257A (en) | Microbial bacterium for degrading long-chain alkane and application thereof | |
| CN108102979A (en) | A kind of degradation bacteria strains JN5 of oily sludge petrochina hydro carbons and its application | |
| CN111733098B (en) | Application of bacillus in low-temperature degradation of petroleum hydrocarbon | |
| CN102250794A (en) | Pseudoxanthomonasjaponensis and microorganism microbial inoculum as well as applications thereof | |
| CN107629976A (en) | A kind of microorganism and its microbial inoculum and application | |
| CN109486726A (en) | The bacterial strain of one plant of degradable petroleum hydrocarbon and its application | |
| CN101935631B (en) | Ralstoniasp. and application thereof in bioremediation of petroleum-contaminated saline-alkali soil | |
| CN108300674A (en) | A kind of oil degradation bacteria and its preparation method and the application in degrading crude oil | |
| CN108034626A (en) | A kind of degradation bacteria strains JN1 of oily sludge petrochina hydro carbons and its application | |
| CN108102978A (en) | A kind of degradation bacteria strains JN8 of oily sludge petrochina hydro carbons and its application | |
| CN108048374A (en) | A kind of degradation bacteria strains JN4 of oily sludge petrochina hydro carbons and its application | |
| CN104845890B (en) | Applications of earth mould (Agromyces sp.) the MT E in a variety of phthalic acid esters of degrading | |
| CN104805018B (en) | Earth mould (Agromyces sp.) MT E of one plant of a variety of phthalic acid ester that can degrade simultaneously | |
| TWI589694B (en) | Acinetobacter junii ds44 isolate having the benzene and/or naphthalene degrading ability and uses of the same | |
| CN108034625A (en) | A kind of degradation bacteria strains JN7 of oily sludge petrochina hydro carbons and its application | |
| CN104046580A (en) | Sphingobacterium strain for degrading polycyclic aromatic hydrocarbon organic pollutant and application thereof | |
| CN103614324B (en) | Short-chain fatty acid degradation bacteria 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 | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20180126 |
|
| WD01 | Invention patent application deemed withdrawn after publication |