CN101935630A - Rhodococcus equi strain and use thereof in petroleum microorganism yield increase - Google Patents
Rhodococcus equi strain and use thereof in petroleum microorganism yield increase Download PDFInfo
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- CN101935630A CN101935630A CN2010102159828A CN201010215982A CN101935630A CN 101935630 A CN101935630 A CN 101935630A CN 2010102159828 A CN2010102159828 A CN 2010102159828A CN 201010215982 A CN201010215982 A CN 201010215982A CN 101935630 A CN101935630 A CN 101935630A
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Abstract
The invention discloses a Rhodococcus equi strain and use thereof in petroleum microorganism yield increase and discloses a method for degrading hydrocarbons in crude oil and producing biosurfactants by using Rhodococcus equi. The method is characterized in that: when the Rhodococcus equi is used to degrade the hydrocarbons in crude oil, the crude oil degradation rate is up to 60 percent and the solid paraffin degradation rate is up to 30 percent; and the glycolipid biosurfactants produced in the metabolism of the Rhodococcus equi can reduce the surface tension of the fermentation liquor to less than 30mN/m.
Description
Technical field
The present invention relates to crude oil hydro carbons mass degradation, a strain Rhodococcus equi and the application in petroleum microorganism volume increase thereof specifically utilizes the method that hydrocarbons is produced the glycolipid type biological surfactant in the strains for degrading crude oil of Rhodococcus equi.
Background technology
In recent years, in large scale mining, processing and the transportation of oil, the whole world has nearly ten million ton of crude oil to enter environment as pollutent, polluted-water, soil and atmosphere every year.Petroleum pollution has become one of worldwide main public hazards.Biological degradation is the important channel that nature is eliminated this pollutant.According to statistics, up to now, have been found that can degraded oil microorganism kind more than 200 is arranged, wherein bacterium has Rhodopseudomonas (Pseudomonas sp.), Corynebacterium (Corynebacteium sp.), micrococcus sp (Micrococcus sp.), Alkaligenes (Alcaligenes sp.) etc.The microorganisms tensio-active agent is one of mechanism of its metabolism crude oil.Bio-surfactant has emulsified crude oil, biodegradable advantage, in the reparation of crude oil pollution soil, has very high using value, so efficiently produce the extensive concern that the screening of the bacterial strain of tensio-active agent and degrading crude oil has caused Chinese scholars.
Summary of the invention
The present invention relates to a kind of method of utilizing the strains for degrading crude oil hydro carbons material production glycolipid type biological surfactant of Rhodococcus equi, specific embodiments is:
One strain Rhodococcus equi, be Rhodococcus equi BS001, classification name: Rhodococcus equi Rhodococcus equi., be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number: CGMCC No3885, preservation date on 06 01st, 2010, it has degradation capability to hydro carbons in the crude oil, but and metabolism generation glycolipid type biological surfactant.Bacterial strain uses therefor is a Rhodococcus equi, and bacterium source is served as reasons and picked up from enrichment screening gained in the Daqing oil field oil-polluted soils sample.
1) preparation of seed liquor: adopt enrichment medium that Rhodococcus equi is carried out enrichment culture, culture medium prescription is: peptone 5-15g/L, yeast powder 2-8g/L, NaCl 2-8g/L, pH value 6.0-8.0; Culture temperature is 30-60 ℃;
2) preparation of fermentation liquid: the Rhodococcus equi fermented liquid that enrichment is obtained is transferred and is cultivated in the crude oil substratum, and the crude oil culture medium prescription is: NH
4Cl 1-5g/L, K
2HPO
41-3g/L, KH
2PO
41-5g/L, CaCl
20.1-1g/L, crude oil 0.5-5g/L; Culture temperature is 30-60 ℃, and incubation time is 3-10 days, and shaking speed is 100-200rpm, and inoculum size is 3-10%;
3) alkane degradation effect Analysis: utilize vapor-phase chromatography that the oil degradation effect is analyzed, used gas-chromatography is that condition is: 100 ℃ of constant temperature 2min are that 4 ℃/min is warmed up to 300 ℃ with temperature rise rate, and then constant temperature 20min, carrier gas N
2Flow velocity 1-2mL/min, H
2Flow velocity 35mL/min, airflow 400mL/min, make-up gas (N
2) flow velocity 26mL/min.
4) preparation of bio-surfactant: use chloroform and methyl alcohol (2: 1～3.5: 1) as extraction agent above-mentioned fermented liquid, according to fermented liquid: the ratio of extraction agent=3: 1～4: 1 extracts fermented liquid, continuous extraction merges organic phase 3 times, evaporated under reduced pressure obtains pale brown toner powder object under 50 ℃ of conditions, is the tensio-active agent crude product.
Adopt Fourier infrared spectrograph and mass spectrum to analyze above-mentioned tensio-active agent crude product, Fourier infrared spectrograph adopts the German Brooker spectral instrument production TENSOR27 of company Fourier infrared spectrograph.
LC-MS adopts: HP1100 type LC-MS instrument, and A phase pure water, B phase acetonitrile, A/B=90/10 kept 3 minutes, and A/B reaches 10/90 when reaching 33 minutes, and when reaching 63 minutes, A/B reaches 0/100, reaches 73 minutes and stops.The C18 chromatographic column detects wavelength 254nm.Mass spectrum is an electric spray ion source, single level Four bar mass analyzer, operating parameter is: dry gas flow velocity (drying gas) 8L/min, spray chamber pressure (nebulizer pressure) 40psig, 350 ℃ of dry gas temperature (drying gas temperature), capillary voltage (capillary voltage) 3000v, CID voltage 70v in the source, sweep limit 190-1000atmu.Characteristic ion: ES (-): [M-H]-[M+Cl].
Description of drawings
Fig. 1 bacterial strain BS001 systematic evolution tree;
Fig. 2 bacterial strain BS001 fermented liquid surface tension and its growth conditions relation;
Fig. 3 bacterial strain BS001 produces thing tensio-active agent infrared spectrogram;
Fig. 4 tensio-active agent high performance liquid phase figure (wherein yellow redness is the rhamnolipid standard substance, and green is that bacterial strain BS001 produces tensio-active agent);
The former petroleum paraffin degradation efficiency of Fig. 5 bacterial strain BS001 and its growth conditions relation;
Crude oil total hydrocarbon scanning spectra before and after Fig. 6 bacterial strain BS001 handles.
Rhodococcus equi BS001, classification name: Rhodococcus equi Rhodococcus equi., be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number: CGMCC No3885, preservation date on 06 01st, 2010, it has degradation capability to the crude oil hydrocarbons, but and metabolism generation glycolipid type biological surfactant.
Embodiment
Embodiment 1: the present invention screens a kind of bacterial classification that produces glycolipid class tensio-active agent from the crude oil contaminated samples, and the chamber is identified pure bacterial strain and produced glycolipid and characterizes by experiment.Realize in the following manner:
1. be enrichment medium with LB substratum (peptone 10g/L, yeast powder 5g/L, NaCl 5g/L, pH value 7.0-7.2), Rhodococcus equi in the sample is carried out enrichment.The line separation of carrying out repeatedly with plate streak is viewed as pure bacterial strain until electron-microscope scanning, and 16S rDNA sequencing analysis is entrusted Takara (Dalian) company.Utilize the NCBI geneseq database to do the comparative analysis of BLAST sequence, choose the higher sequence of homology and adopt Mega4 software according to ortho position connection method constructing system evolutionary tree (as shown in Figure 1).
Bacterial strain is Rhodococcus equi BS001, classification name: Rhodococcus equi Rhodococcus equi., be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number: CGMCCNo3885, preservation date on 06 01st, 2010, it has degradation capability to the crude oil hydro carbons, but and metabolism generation glycolipid type biological surfactant.
2. bacterial strain BS001 is placed under differing temps, salinity and the pH value condition respectively and cultivate, observe the strain growth top condition.Through optimizing bacterial strain BS001 tolerance pH scope is 4.0～9.0,40～55 ℃ of temperature, salinity 0.1%～8%; Optimal growth condition is 40 ℃ of temperature, pH=7.0, salinity 2%; Surface tension is minimum to be 29.3mN/m.
3. the concrete fermenting process of bacterial strain BS001: press the 3-10% inoculation of substratum quality, fermentative medium formula is: NH
4Cl 1-5g/L, K
2HPO
41-3g/L, KH
2PO
41-5g/L, CaCl
20.1-1g/L, crude oil 0.5-5g/L.Culture temperature is 30-60 ℃, and incubation time is 3-10 days, and shaking speed is 100-200rpm, gets fermented liquid.
The surface tension of measuring fermented liquid after fermentation is finished drops to 30.1mN/m from 61.0mN/m.Get BS001 fermented liquid 8000rpm, centrifugal 20min removes thalline, chloroform and methyl alcohol (2: 1～3.5: 1) are as extraction agent, according to fermented liquid: the ratio of extraction agent=3: 1～4: 1 extracts fermented liquid, continuous extraction merges organic phase 3 times, evaporated under reduced pressure obtains pale brown toner powder object under 50 ℃ of conditions, is the tensio-active agent crude product.By the infrared spectra mass spectroscopy, the bio-surfactant that preliminary judgement produces is a glycolipid compound.
Shown in Fig. 2-4: Fig. 2 bacterial strain BS001 fermented liquid surface tension and its growth conditions relation; Fig. 3 bacterial strain BS001 produces thing tensio-active agent infrared spectrogram; Fig. 4 tensio-active agent high performance liquid phase figure (wherein yellow redness is the rhamnolipid standard substance, and green is that bacterial strain BS001 produces tensio-active agent);
Characterize by infrared and LC-MS, determine that the bacterial strain that rhodococcus produces that screening obtains is a rhamnosyl lipid tensio-active agent.The low energy of fermented liquid surface tension is reduced to 29 left and right sides mN/m from 73mN/m.
Embodiment 2: screening is obtained can degrading crude oil and the bacterial strain BS001 of paraffin, investigates its degradation rate to hydro carbons in the crude oil with n-dodecane for the feature carbon source; With crude oil and paraffin is that carbon source is investigated its degradation rate and time relation.Realize by following scheme in the laboratory:
1. be configured to the substratum that n-dodecane is independent carbon source, it is as follows to fill a prescription: n-dodecane 1%～3%; NH
4NO
32g/L; K
2HPO
41.5g/L; KH
2PO
43g/L; CaCl
20.1g/L; MgSO
40.1g/L the pH value transfers to 6.0-8.0,121 ℃ of autoclaving 20min.
Obtain bacterial strain BS001 seed liquor by screening among 1%～5% inoculum size inoculation embodiment 1 of substratum quality, specifically consist of peptone 5-15g/L, yeast powder 2-8g/L, NaCl 2-8g/L, pH value 6.0-8.0; Fermentation condition is: 30～37 ℃ of temperature, rotating speed 150～200rpm, time: 36～48h, shaking table aerobic fermentation.After fermentation is finished is 46.2% with spectrophotometry n-dodecane degradation rate.
It shows that to being that the degradation rate of the normal paraffin of representative reaches 46.2% with the n-dodecane it has good degradation rate to normal paraffin as can be seen by experiment.
2. be configured to the substratum that crude oil or paraffin are independent carbon source, it is as follows to fill a prescription: crude oil or paraffin 1%～3%; NH
4NO
32g/L; K
2HPO
41.5g/L; KH
2PO
43g/L; CaCl
20.1g/L; MgSO
40.1g/L the pH value transfers to 6.0-8.0,121 ℃ of autoclaving 20min.
Obtain bacterial strain BS001 by screening among 1%～5% inoculum size inoculation embodiment 1 of substratum quality, fermentation condition is: 30～37 ℃ of temperature, rotating speed 150～200rpm, time: 36～48h, shaking table aerobic fermentation.After finishing, fermentation uses the spectrophotometry degradation rate, with its fermented liquid OD
600Characterize its thalli growth situation.
As shown in Figure 5, the former petroleum paraffin degradation efficiency of Fig. 5 bacterial strain BS001 and its growth conditions relation;
By experiment as can be seen, bacterial strain reaches about 60% the oil degradation rate, and the paraffin degradation rate is reached 37%, and oil degradation concentrates on the strain growth logarithmic phase, and the paraffin degraded concentrates on bacterial strain stationary phase.Bacterial strain all has certain effect to crude oil and paraffin degraded.
Embodiment 3: screening obtains the pure bacterial strain of each normal paraffin in the extensive degrading crude oil.Realize by following scheme in the laboratory:
Be configured to the substratum that crude oil is independent carbon source, it is as follows to fill a prescription: crude oil 1%～3%; NH
4NO
32g/L; K
2HPO
41.5g/L; KH
2PO
43g/L; CaCl
20.1g/L; MgSO
40.1g/L the pH value transfers to 6.0-8.0,121 ℃ of autoclaving 20min.
Obtain bacterial strain BS001 seed liquor by screening among 1%～5% inoculum size inoculation embodiment 1 of substratum quality, specifically consist of peptone 5-15g/L, yeast powder 2-8g/L, NaCl 2-8g/L, pH value 6.0-8.0; Fermentation condition is: 30～37 ℃ of temperature, rotating speed 150～200rpm, time: 36～48h, shaking table aerobic fermentation.With the irreducible oil in the n-hexane extraction degraded secondary fermentation liquid, with gas-chromatography it being done the scanning of total hydrocarbon component according to the method in " SY-T 5779-1995 crude oil total hydrocarbon gas chromatography analysis method " after fermentation is finished, is contrast with normal crude oil.
From gas chromatogram (as shown in Figure 6) as can be seen, good degradation effect is arranged all from the normal heptane to the n-tetracosane, the normal heptane in the crude oil reaches more than 95% to the n-dodecane degradation rate.Its normal paraffin to each component of crude oil all has good degradation effect as can be seen.
The Rhodococcus equi that screens among the present invention can degrading crude oil and paraffin and the tensio-active agent oil degradation rate that can produce rhamnosyl lipid can reach 60%, the solid paraffin degradation rate can reach 30%, the glycolipid type biological surfactant that its metabolism produces can make the surface tension of fermented liquid be reduced to below the 30mN/m.This bacterial strain has the prospect of well utilizing in the microbe oil production field.
Claims (7)
1. a strain Rhodococcus equi, the bacterial classification that is adopted is Rhodococcus equi BS001, classification name: Rhodococcus equi Rhodococcus equi., be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number: CGMCC 3885, preservation date on 06 01st, 2010.
2. the application of the described Rhodococcus equi of claim 1 in the petroleum microorganism volume increase, it is characterized in that: described Rhodococcus equi BS001 has degradation capability to hydrocarbons in the crude oil, but and metabolism generation glycolipid type biological surfactant.
3. application according to claim 2 is characterized in that:
The adoptable enrichment medium of described Rhodococcus equi consists of: peptone 5-15g/L, yeast powder 2-8g/L, NaCl 2-8g/L, pH value 6.0-8.0; Culture temperature is 30-60 ℃.
4. application according to claim 2 is characterized in that:
The described method of Rhodococcus equi degrading crude oil hydrocarbons of utilizing is: adopt the crude oil substratum, culture temperature is 30-60 ℃, and incubation time is 3-10 days, and shaking speed is 100-200rpm, and inoculum size is 3-10%.
5. application according to claim 2 is characterized in that:
The described method of utilizing Rhodococcus equi to produce bio-surfactant is: adopt the crude oil substratum, culture temperature is 40-55 ℃, and incubation time is 1-3 days, and shaking speed is 100-200rpm, and inoculum size is 3-10%;
Cultivate the separation of bio-surfactant in the system of back and adopt organic solvent extractionprocess.
6. method according to claim 4 is characterized in that:
Adopt organic solvent extractionprocess, the concrete operations step is: use 2: 1～3.5: 1 chloroform of volume ratio and methyl alcohol as extraction agent, according to fermented liquid: the volume ratio of extraction agent=3: 1～4: 1 extracts fermented liquid, continuous extraction 2-3 time, merge organic phase, evaporated under reduced pressure obtains pale brown toner powder object under 50 ℃ of conditions, is the tensio-active agent crude product.
7. according to claim 3 or 4 described methods, it is characterized in that:
Described crude oil nutrient media components is: NH
4Cl 1-5g/L, K
2HPO
41-3g/L, KH
2PO
41-5g/L, CaCl
20.1-1g/L, crude oil 0.5-5g/L.
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| CN102732424A (en) * | 2011-04-15 | 2012-10-17 | 大连百奥泰科技有限公司 | Compounded oil extraction microbe and its application in thickened oil and extra thickened oil exploitation |
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| CN108251412A (en) * | 2018-02-11 | 2018-07-06 | 中国科学院寒区旱区环境与工程研究所 | The mutagenesis screening method of Rhodococcus strain YF28-1-4 and application |
| CN114591841A (en) * | 2022-03-02 | 2022-06-07 | 吉林大学 | Rhodococcus equi strain and application thereof in preparation of inactivated vaccine |
| CN114591841B (en) * | 2022-03-02 | 2024-01-30 | 吉林大学 | Rhodococcus equi and application thereof in preparation of inactivated vaccine |
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