CN103834590B - One strain activity Thermophilic Bacteria and application thereof - Google Patents
One strain activity Thermophilic Bacteria and application thereof Download PDFInfo
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- CN103834590B CN103834590B CN201410033904.4A CN201410033904A CN103834590B CN 103834590 B CN103834590 B CN 103834590B CN 201410033904 A CN201410033904 A CN 201410033904A CN 103834590 B CN103834590 B CN 103834590B
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Abstract
The present invention provides a kind of thermophilic microorganism, belongs to XS2 bacterial strain (Geobacillus pallidus) for Bacillus licheniformis, and the preserving number of this bacterial strain is CGMCC No.8338.The XS2 bacterial strain of the present invention is for the outer biological emulsifier of fermenting and producing born of the same parents.Thermophilic Bacteria XS2 in the present invention has the strongest tolerance to pH, temperature, salinity, has and oil polluted environment and reservoir media are adapted to ability widely.The active bio emulsifying agent that strain X S2 produces, there is yield high, activity stabilized, strong adaptability, the advantages such as production cost relative moderate, can apply to microorganism and improve oil recovery, repairing environment pollutes and chemical industry, and this has stronger potentiality to be exploited in terms of the range of application improving China's industrial bio emulsifying agent and efficiency.
Description
Technical field
The invention belongs to functional microorganism screening, applied technical field, be specifically related to strain thermophilus strain and an application thereof, i.e. one strain can produce the bacterial strain of active bio emulsifying agent, and this bacterial strain transforming glucose secretes the application of the outer biological emulsifier of born of the same parents.
Background technology
Surfactant is simultaneously containing hydrophilic and lipophilic group, just can significantly reduce the general name of the amphiphile, amphiphilic molecule compound of solvent surface tension or liquid-liquid interface tension force under very low concentrations.Feature in structure determines the multiple physicochemical property of surfactant, as amphiphilic, dissolubility, surface adsorption etc., is widely used in the fields such as petroleum industry, environmental project, food industry and leather, building, cosmetics.
The molecule of many biogenetic derivations also has hydrophilic and lipophilic group, show very high surface, generally hydrophilic and oleophylic activity having, microorganism, animal or plant the natural surfactant produced is referred to as biosurfactant (Biosurfactants).Biosurfactant has that selectivity is good, consumption is few, nontoxic, degradable, pollution-free, the advantages such as new chemical group can be introduced.The petroleum industries such as biosurfactant, at viscosity reduction, improves oil recovery factor, the biological restoration of oil-polluted soils and field of environment engineering have a wide range of applications.And the microorganism of biosurfactant can be produced due to its extremely strong adaptive capacity to environment, Fast-propagation ability and the high activity of degraded oil pollutant, and it being most widely used in oil pollution reparation, effect is preferably also.At oil pollution area microbe inoculation, making microorganism produce Bio-surface active material, accelerate the emulsifying to hydro carbons decomposing also is the most commonly used method for governing pollution.
Research finds in the microorganism of production biosurfactant, have just forms surfactant when only utilizing the hydrophobicity carbon sources such as hydro carbons in degraded, some microorganisms then only just form surfactant when utilizing water solublity carbon source, and the microorganism also having can synthesising biological surfactant in two class carbon sources.And, the bacterial strain of current biological emulsifier is in the following problem of the existence of application aspect: 1, and activity is unstable, and the most active most strong bacterial strains cannot be applied to actual production；2, adaptive capacity to environment is poor, and actual environment is excessive with laboratory condition difference, as many in reservoir temperature at 30-70 DEG C, and the general condition of culture of laboratory is 25-30 DEG C, cause microorganism cannot in actual environment normal growth and produce activity；3, metabolite yields poorly, and activity is weak, causes at transport and application aspect high cost, and using effect is undesirable.Therefore, the microorganism of the production biosurfactant that screening has practical value just has great importance.
Summary of the invention
It is an object of the invention to provide and a kind of metabolism can produce the thermophilic microorganism bacterial strain of biosurfactant, and this bacterial strain transforming glucose secretes the application of the outer biological emulsifier of born of the same parents, thus make up the deficiencies in the prior art.
Present invention firstly provides a kind of thermophilic microorganism, XS2 bacterial strain (Geobacillus pallidus) is belonged to for Bacillus licheniformis, the preserving number of this bacterial strain was CGMCC No.8338, was positioned at China Committee for Culture Collection of Microorganisms's common micro-organisms center of No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City in preservation on October 15 in 2013.
The XS2 bacterial strain of the present invention is for the outer biological emulsifier of fermenting and producing born of the same parents；
The present invention also provides for a kind of method of fermenting and producing extracellular products, is to carry out the outer biological emulsifier of fermenting and producing born of the same parents with XS2 bacterial strain transforming glucose；
Wherein for the culture medium of fermentation, include the component of following mass percent: glucose 2%, sodium nitrate 0.3%, potassium dihydrogen phosphate 0.042%, dipotassium hydrogen phosphate 0.12%, ferrous sulfate 0.005%, magnesium sulfate 0.05%, calcium chloride 0.005%；Medium pH is 7.5；
Fermentation liquid is mixed in equal-volume methanol mixed, and after-20 DEG C of freeze overnight, 6000rpm is centrifuged 20min, and supernatant discarded obtains emulsifying agent crude product；
The biological emulsifier that the XS2 bacterial strain of present invention screening is produced has the strongest emulsion stability, and fermenation raw liquid is to still keeping 68.6 ± 0.4% after emulsion stability 24h of liquid paraffin；
Thermophilic Bacteria XS2 in the present invention has the strongest tolerance to pH, temperature, salinity, has and oil polluted environment and reservoir media are adapted to ability widely.The active bio emulsifying agent that strain X S2 produces, there is yield high, activity stabilized, strong adaptability, the advantages such as production cost relative moderate, can apply to microorganism and improve oil recovery, repairing environment pollutes and chemical industry, and this has stronger potentiality to be exploited in terms of the range of application improving China's industrial bio emulsifying agent and efficiency.
Accompanying drawing explanation
Fig. 1: carbon source affects figure to bacterial strain metabolism synthesising biological emulsifying agent of the present invention；
Wherein: 1-fructose 2-galactose 3-sucrose 4-maltose 5-isobutyltrimethylmethane. 6-edible oil 7-arabinose 8-glucose 9-rhamnose 10-liquid paraffin 11-lactose 12-soybean lecithin 13-starch 14-chitosan；
Fig. 2: nitrogen source affects figure to bacterial strain metabolism synthesising biological emulsifying agent of the present invention；
Fig. 3: each Parameter Variation figure in emulsifying agent production process；
Fig. 4: fed-batch fermentation cell concentration and emulsifying agent change of production figure；
Fig. 5: different spaces core simulated fermentation broth improves oil recovery Parameters variation figure.
Detailed description of the invention
Below in conjunction with embodiment, the bacterial strain of the present invention is described in detail.
Embodiment 1: the screening of biosurfactant-producing microbe strain
The living features of thermophilic microorganism depends on the particularity of its growing environment, the physiological saline solution vibration mixing with 5 times of volumes of the Gansu, China Yumen Oilfield duck gorge oil reservoir oil-polluted soils sample will be taken from, take 100 μ l soil suspension applying solid LB flat boards respectively, 3d cultivated by incubator 60 DEG C, picking monoclonal, again isolated and purified at the flat lining out of LB, until obtaining monoclonal after purification, finding through Gram’s staining, strain X S2 is gram positive bacteria；Through microscope it has been observed that strain X S2 is rod-short, there is spore；Semi-solid percutaneous puncture-inoculation experiment shows, strain X S2 is amphimicrobian, and nitrate reduction and hydrolysis of lipid experimental result are positive.Extract cell DNA, with 27F (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and 1387R (5 '-GGGCGGWGTGTACAAGGC-3 ') primer PCR amplification 16S rRNA gene, find after order-checking comparison, strain X S2 is the highest with the sequence homology of Geobacillus pallidus, identifies that this bacterial strain is that ground bacillus belongs to.Cultivation shows, strain X S2 can utilize various saccharides (glucose, sucrose, fructose) and hydrocarbons (paraffin, glycerol, hexadecane, crude oil) galactopoiesis agent, this bacterial strain is at 40-80 DEG C, Nacl concentration 5-20% (w/v), all can journey grow under the conditions of pH5-9, degrading crude oil (hydro carbons of C9-C37) activity is strong, produce biological emulsifier activity height and (tolerate 40-80 DEG C, pH5-12, Nacl concentration 1-20%, it is more than 68% to liquid paraffin emulsifying capacity E24), optimum growth temperature is 60 DEG C, under the conditions of 3d is cultivated in shaking table 180rpm concussion, bacterial strain reaches 45.8% to oil degradation rate, i.e. 9.16g/L.Strain X S2 delivers to China General Microbiological culture presevation administrative center (CGMCC) preservation, and preserving number is CGMCC No.8338.
Embodiment 2: the activity of bacterial strain Geobacillus sp.XS2 and emulsifying agent performance
1, prepared by bacterium solution: seed liquor coating LB flat board, the preparation of LB culture medium is following (w/v): peptone 1%, yeast extract 0.5%, sodium chloride 1%, solid medium adds 2% agar powder, pH7.5, is inverted in overnight incubation in 60 DEG C of constant incubators.Choose single bacterium colony, be scoring on fresh LB flat board, repeat 2～3 times to obtain pure bacterium.By inoculation in 200ml contains the 500mL triangular flask of 2% (w/v) crude oil minimal medium, 180rpm, 60 DEG C of shaken cultivation, inorganic salt formula is as follows: glucose 2%, sodium nitrate 0.3%, potassium dihydrogen phosphate 0.042%, dipotassium hydrogen phosphate 0.12%, ferrous sulfate 0.005%, magnesium sulfate 0.05%, calcium chloride 0.005%, medium pH 7.5.Crude oil culture medium is that the glucose in above-mentioned minimal medium replaces with crude oil, is used for measuring strain X S2 degrading crude oil activity.Fermentation system is at 60 DEG C, and under the conditions of 3d is cultivated in shaking table 180rpm concussion, number of viable is up to 1010Individual/ml.
2, fermentation liquid emulsifying activity and oil degradation activity
By equal-volume fermentation liquid with the emulsifying activity of liquid paraffin vortex oscillation hybrid test fermentation liquid, result shows, under the conditions of fermentor cultivation 40h, fermenation raw liquid reaches 68.6 ± 0.4% in 24 hours to the emulsion stability of liquid paraffin, shows stronger emulsifying activity.
Strain X S2 seed is inoculated in 2% (w/v) crude oil minimal medium, 60 DEG C, 3d is cultivated in shaking table 180rpm concussion, equal-volume n-hexane extraction residual oil, gas chromatographic analysis bacterial strain is to oil degradation scope and degradation rate, it was demonstrated that the hydro carbons of C8-C18 in bacterial strain Geobacillus sp.XS2 obvious degradation crude oil, and crude oil high degradation rate is 45.8%, i.e. 9.16g/L, shows stronger oil degradation activity.
3, emulsifying agent separates and performance
By fermentation liquid at 4 DEG C, under the conditions of 10000rpm, centrifugal 30min removes thalline, Rotary Evaporators in 5: 1 ratio concentrated supernatant, transfer in beaker, adding triploid accumulation of pathogenic cold methanol solution (-20 DEG C of pre-cooling 30min in advance), 4 DEG C stand overnight, 11000rpm, 4 DEG C of centrifugal 25min collect precipitation, are biological emulsifier crude product.
Use gel permeation chromatography (Gel Permeation Chromatography, GPC) to analyze biological emulsifier to cause, containing saccharide 69.6% in biological emulsifier, containing lipid 22.7%, containing protein-based 8.7%.Can tolerate 30-60 DEG C of temperature, the pH scope of 3-12 and the salinity of 1-20% (w/v), this feature is especially suitable for the high salinity of China, high pH and the Microbial Enhanced Oil Recovery of high-temperature oil reservoir and oil pollution reparation.
Embodiment 3: bacterial strain Geobaciuus sp.XS2 the most adaptable method optimizes
1, carbon nitrogen source screening.Strain X S2 can utilize the several kinds of carbon source growths such as sugar, organic acid, alcohol, fat, hydrocarbon.When utilizing glucose to do carbon source, cell growth rate and emulsifying activity all reach the highest, and emulsifying agent yield is the highest simultaneously, is illustrated in fig. 1 shown below.Having screened 5 kinds of nitrogen sources to be illustrated in fig. 2 shown below the impact of thalline XS2 synthetic emulsifier, when nitrogen source is done in sodium nitrate, fermentation liquid emulsifying activity and emulsifying agent yield are the highest simultaneously.Therefore, select glucose and sodium nitrate as the optimal carbon nitrogen source of biological emulsifier fermenting and producing.
Study proof further, under 2% glucose condition, when nitrogen concentration be 3g/L, i.e. carbon-nitrogen ratio be 20: 3 time, cell concentration is maximum；When nitrogen concentration is 5g/L, and carbon-nitrogen ratio is 20: 4, the yield of biological emulsifier is the highest.Therefore, when XS2 strain fermentation produces emulsifying agent, use fed-batch fermentation pattern, control the carbon-nitrogen ratio in sweat each stage, reached the optimum state of thalli growth and Product formation.
2, fermentation condition optimization and fermentation kinetics research
2.1 training systern.With fermentation liquid OD, E24 and emulsifying agent yield as index, optimize strain X S2 inoculum concentration in fermentation tank, initial pH, speed of agitator and the ventilation impact on sweat.Result proves when inoculum concentration is 10% (v/v), and pH is between 7-7.5, and ventilation is 4L/min, and when speed of agitator is 550rpm, cell density and emulsifying agent yield reach the highest (Fig. 3).Experiment proves that in the application, biological emulsifier is typical some growth coupled model, and the biological emulsifier being ultimately formed is made up of two parts, the large number of biological emulsifying agent that the biological emulsifier synthesized including the thalli growth stage and thalli growth synthesize after stopping.Conclusion is optimal feed methods and develops new fermentation technology, improves biological emulsifier fermentation level further and provides theoretical direction.
Fed-batch fermentation is carried out respectively at cultivation 8h, 16h, 24h and 32h.Feed supplement liquid is 6% (w/v) NaNO3With 40% (w/v) glucose solution, add 100ml NaNO every time3Solution and 200ml glucose solution.As shown in Figure 4, through adding carbon and nitrogen sources, improve the final concentration of thalline, the yield of biological emulsifier is greatly improved the most therewith, up to 23.4g/L, is about 5 times of initial emulsifying agent yield.
Embodiment 4: bacterial strain Geobacillus sp.XS2 is improving the application of oil recovery (MEOR)
Have studied biosurfactant pilot scale culture; as embodiment 2 is stated; small-sized fermentation tank is used to complete lab scale research in laboratory and run; further biosurfactant application in tertiary oil recovery is carried out deep checking; implementation process is as follows: utilize the core Oil Displacing Capacity at physical condition Imitating biosurfactant of four kinds of different aperture degree and permeability; complication experiment is carried out in conjunction with the crude oil of two kinds of different viscosities and the polyacrylamide (HPAM) of 0.15%, as shown in table 1 below.
Table 1: biosurfactant physical contradictions is studied
Relation between pressure, moisture content and recovery ratio is as shown in Figure 5 in simulation experiment for four kinds of different cores, according to experimental data, after deduction water drive effect, it is calculated fermentation liquid raising oil recovery and respectively reaches 11.55%, 15.16%, 21.51% and 30.78%, hence it is evident that improve average level 3-5 times of oil recovery 3-5% higher than microorganism, illustrate that this system has preferable application potential, the recovery ratio of remaining oil after water drive can be increased substantially.
Embodiment 5: bacterial strain Geobacillus sp.XS2 is processing petroleum-contaminated soil and the application of oil-containing discarded slurry
Further the fermentation liquid containing emulsifying agent is used for oil-polluted soils and oil-containing discarded slurry reparation, finds that there is preferable effect.
1, the emulsifying agent repair to petroleum-contaminated soil
Cultured strain X S2 fermentation liquid, add 10% (w/v) petroleum-contaminated soil (petroleum hydrocarbon content more than 5%), shaking table 30 DEG C, 180rpm shaken cultivation 3d, find that crude oil is fermented that liquid is dispersed and emulsifying, can be seen that emulsifying agent is fine to the emulsifying degradation effect of oily sludge, the speed of biological restoration can be accelerated, improve remediation efficiency.
Owing to the biological restoration process of the soil of oil pollution is complicated, it recovers soil fertility completely and production capacity needs the longer time, in order to accelerate reparation speed and the quality of microorganism and emulsifying agent, Dagang Oilfield oil-polluted soils plot is marked 500 square meters and is carried out preliminary treatment by seminar, greatly reduce total petroleum hydrocarbons content, in 30 days, the degradation efficiency of total petroleum hydrocarbon reaches more than 60%, and in 60 days, the degradation efficiency of total petroleum hydrocarbon reaches more than 75%.Through the continuous experiment of nearly 2 years, substantially the production capacity of soil has been recovered, First Year reduces the petroleum hydrocarbon in IA and salinity respectively reaches 90% and more than 70%, progressively recover the growth of the saline alkali tolerant plant such as Suaeda heteroptera, Radix seu Caulis Embeliae Parviflorae, through the microbial treatments of continuous 3 years, in conjunction with measures such as rainwater drip washing, in the 3rd year, can grow fine this plot maize planting crop.
2, the emulsifying agent repair to oil-containing discarded slurry
Waste drilling mud is the mixed waste of the oil-water-mud produced during oilfield drilling, they and sump oil, sewage, the three big pollutant that oil field produces are collectively constituted, mainly include rejected well drilling liquid, waste liquid, drilling cuttings etc., its granule tiny (0.01-0.3um), water content is high (30-90%), generally in alkalescence (pH8-12), semifluid form, mainly contain heavy metal, oils, bentonite, alkali compounds etc., such as chromium-iron lignosulfonate, sulfonated gilsonite, sulfonated lignites etc. are all to people, poultry and the material of bad environmental, cause pH too high, water body and the oil of soil, heavy metal pollution.
Analyze the analysis of physical and chemical property of Dagang Oilfield high salinity discarded slurry, determining that its main pollutional condition is that saline alkali is petroleum hydrocarbon contaminated, pollutant are mainly the hardly degraded organic substances such as aromatic hydrocarbon (42.01%), asphalitine (20.31%) and colloid (18.92%).Emulsifying agent is as follows to the processing procedure of discarded slurry: first pass through the processing mode of embodiment 2, obtain the high cell density fermentation of strain X S2 galactopoiesis agent, cultivate containing emulsifying agent fermentation liquid 100L on a small scale, by cultured strain X S2 fermentation liquid, ratio according to 1: 1 mixes (petroleum hydrocarbon content 1.5-2%) with discarded slurry, shaking table 30 DEG C, 180rpm shaken cultivation 30d, finds that the dispersed emulsifying of liquid that is fermented in mud becomes the drop of naked eyes invisible (diameter is less than 100um).
N-hexane extraction emulsifying agent processes oil-containing discarded slurry, mensuration obtains petroleum hydrocarbon degradation rate and reaches 55.13%, wherein the degradation rate of alkane is the highest, reach 92.04%, aromatic hydrocarbon degradation rate is 79.5%, the degradation rate of resin and asphalt is respectively 10.27% and 16.46%, it is shown that adaptive capacity to environment that emulsifying agent is stronger and activity.
Claims (1)
1. the application of a biological emulsifier producing strains, it is characterised in that described biological emulsifier producing strains is applied to improve oil and adopts
Yield, repairs petroleum hydrocarbon contaminated soil and water body, and processes waste drilling mud；
Described biological emulsifier producing strains is thermophilic microorganism Geobacillussp.XS2, and preserving number is CGMCC No.8338；
In the fermentation production process of described biological emulsifier, culture medium comprises following mass percent component: glucose 2%, sodium nitrate
0.3%, potassium dihydrogen phosphate 0.042%, dipotassium hydrogen phosphate 0.12%, ferrous sulfate 0.005%, magnesium sulfate 0.05%, chlorine
Change calcium 0.005%；
Working condition is: inoculum concentration is the 10% of fermentating liquid volume, and pH is 7.5, and ventilation is 4L/min, and speed of agitator is
550rpm；
Described fermentation uses fed-batch fermentation；Fed-batch fermentation is carried out respectively at cultivation 8h, 16h, 24h and 32h.
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| CN104152388B (en) * | 2014-08-23 | 2016-07-06 | 中国科学院天津工业生物技术研究所 | One strain activity Thermophilic Bacteria mutant and the application in the displacement of reservoir oil thereof |
| GB201501408D0 (en) * | 2015-01-28 | 2015-03-11 | Statoil Petroleum As | Enhanced oil recovery and environmental remediation |
| CN105441351B (en) * | 2015-07-21 | 2018-08-10 | 南开大学 | One plant of thermophilic production surfactant oil degradation bacteria and its application |
| CN105505827B (en) * | 2016-01-11 | 2018-07-31 | 天津微锐生物科技有限公司 | The gene and application of petroleum sweetening denitrogenation bacterial strain and its desulfurization removing nitric |
| CN106749565B (en) * | 2017-01-05 | 2019-12-10 | 中国科学院天津工业生物技术研究所 | Emulsified protein and application thereof |
| CN106589081B (en) * | 2017-01-05 | 2020-09-18 | 中国科学院天津工业生物技术研究所 | Protein with emulsifying activity and application thereof |
| CN109401974A (en) * | 2018-12-05 | 2019-03-01 | 河北省地质环境监测院 | A kind of oil-polluted soils microbial treatment method |
| CN110628437A (en) * | 2019-10-08 | 2019-12-31 | 北京泷涛环境修复有限公司 | Mixed preparation for repairing petroleum-polluted soil |
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| CN102703348B (en) * | 2012-05-25 | 2013-08-21 | 武汉科技大学 | Alkane degrading bacteria and application thereof |
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| 响应面法优化土芽孢杆菌XS2产生物乳化剂的研究;徐爽 等;《安徽农业科学》;20111231;第39卷(第16期);9768－9769，9788 * |
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