CN107129943B

CN107129943B - Dendrolimus arborescens strain and application thereof

Info

Publication number: CN107129943B
Application number: CN201710278513.2A
Authority: CN
Inventors: 高卉; 薛泉宏; 来航线
Original assignee: Shaanxi Botai Bioengineering Co ltd; Northwest A&F University
Current assignee: Shaanxi Botai Bioengineering Co ltd; Northwest A&F University
Priority date: 2017-04-25
Filing date: 2017-04-25
Publication date: 2020-11-03
Anticipated expiration: 2037-04-25
Also published as: CN107129943A

Abstract

The invention discloses 1 strain of Nectria arborescens, in particular to Dietzia cereidiphili X9, which is preserved in China center for type culture Collection in 2016, 3, 14 days, and has the preservation number of CCTCC M2016120. Also disclosed is its use in microbial enhanced oil recovery and in the bioconversion of crude oil, bitumen, aromatics and gum degradation. The Dietzia cercidiphylli X9 provided by the invention can grow by taking crude oil as a unique carbon source, has strong degradation capability on asphalt, can degrade and convert macromolecular components in the crude oil, such as the asphalt, and the like into light components, such as saturated hydrocarbon, and the like, changes the physicochemical properties of the crude oil, reduces the adhesiveness and the viscosity of the crude oil, can be used for microbial enhanced oil recovery and can be used for biologically converting heavy components in the crude oil into light components, and the yield of the light oil in one-time processing of the crude oil is improved.

Description

Dendrolimus arborescens strain and application thereof

Technical Field

The invention relates to the technical field of microbial enhanced oil recovery and biotransformation, in particular to a strain of Nectria androsaceus and application thereof.

Background

It has been investigated that crude oil near 2/3 in the reservoir is difficult to recover due to its viscous nature. Asphalt is one of the important components determining the viscosity of crude oil, has high content in the thick oil and complex structure, and is extremely difficult to be degraded by microorganisms. The asphalt degradation can greatly reduce the viscosity of crude oil and improve the recovery ratio. The method has the advantages of screening bacteria which have strong asphalt degradation effect and simultaneously produce surface activity, and having important significance for microbial enhanced oil recovery of heavy oil reservoirs with high asphalt content, degrading and converting asphalt in crude oil into micromolecule hydrocarbons such as saturated hydrocarbon and the like, and improving the yield of light oil in one-time processing of the crude oil.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide the Nectria androsaceus strain and the application thereof, wherein the Nectria androsaceus strain can grow by taking crude oil as a unique carbon source, has stronger degradation capability on asphalt, can change the physical and chemical properties of the crude oil, reduces the adhesiveness and viscosity of the crude oil, and has the functions of strengthening oil extraction and degrading and converting heavy components in the crude oil into light components.

In order to achieve the purpose, the invention is realized by adopting the following technical scheme.

One strain of Nectria androsaceus, specifically Dietzia cereidiphilia X9, is deposited in China center for type culture Collection at 3 months and 14 days 2016, and has a preservation number of CCTCC M2016120.

Application of Dietzia cercidiphylli X9 bacterium

(1) Application of Dietzia cercidiphylli X9 strain in asphalt degradation.

(2) Use of the bacterium Dietzia cercidiphylli X9 for the degradation of pectin.

(3) Use of Dietzia cercidiphylli X9 bacterium for the degradation of aromatic hydrocarbons.

(4) Use of the Dietzia cercidiphylli X9 strain in crude oil degradation.

(5) Application of Dietzia cercidiphylli X9 strain in microbial enhanced oil recovery.

(6) Use of Dietzia cercidiphylli X9 in the primary processing of crude oil.

Compared with the prior art, the invention has the beneficial effects that:

the Dietzia cercidiphylli X9 provided by the invention can grow by taking crude oil as a unique carbon source, has stronger degradation capability on asphalt, can degrade macromolecular components such as asphalt in the crude oil into micromolecular components, reduces the adhesiveness and viscosity of the crude oil, can reduce the oil-water interfacial tension by using surface active substances generated by the Dietzia cercidiphylli X9, changes the adhesion capability and the fluidity of the crude oil, changes the physicochemical properties of the crude oil, greatly improves the oil displacement rate, and has important application value on MEOR and the improvement of the content of light components such as saturated hydrocarbon in the crude oil.

Drawings

The invention is described in further detail below with reference to the figures and specific embodiments.

FIG. 1 is a colony morphology map of Dietzia cercidiphylli X9;

FIG. 2 is a cell morphology map (2000-fold magnification) of Dietzia cercidiphylli X9;

FIG. 3 is a cell morphology map (25000 magnification) of Dietzia cercidiphylli X9;

FIG. 4 is a diagram showing the distribution and morphology of bitumen on a pure bitumen slide after being acted upon by Dietzia cercidiphylli X9; wherein, a is amplified by 2 times (reflected light), b is amplified by 20 times (transmitted light), and c is amplified by 40 times (transmitted light); the black part is asphalt, and the white part is transparent spots without asphalt;

FIG. 5 is a graph showing the residual crude oil on the filter paper after treating the crude oil filter paper with Dietzia cercidiphylli X9.

FIG. 6 is a diagram of a device for displacing oil when Dietzia cercidiphylli X9 fermentation broth displaces crude oil.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention.

The invention provides a Dietzia cercidiphylli X9 strain, which is preserved in China center for type culture collection (CCTCC M2016120) 3.14.2016, and the preservation unit address is Wuchang Loa Jia mountain, Wuhan City, Hubei province, China, and the post code 430072.

The materials used in this example are specifically as follows:

bacteria isolation source: the extended oil field of Shaanbei China has 6 groups of oil samples and oil soil beside the oil well.

Crude oil filter paper: dipping a qualitative filter paper with the diameter of 8.5cm into the crude oil, placing the dipped qualitative filter paper into a culture dish with the diameter of 9cm, and sterilizing the culture dish for 30min at 121 ℃.

Separating a culture medium: 10g of agar powder, 5.0g of NaCl, 1L of water, sterilizing for 30min at 121 ℃, pouring into a dish, and tightly attaching the sterilized crude oil filter paper to the surface of the culture medium after the culture medium is solidified.

Strain purification, liquid fermentation and culture medium preservation: beef extract peptone agar medium.

Primary screening of culture medium: KNO₃5.0g，MgSO₄·7H₂O 0.5g，KH₂PO₄1.0g, 1000mL of water, neutral pH value, 10.0g of agar powder and 20.0g of crude oil, sterilizing at 121 ℃ for 30min, shaking up and pouring into a dish.

Re-screening the culture medium: NaNO₃2.0g，MgSO₄·7H₂O 0.5g，KH₂PO₄2.0g，(NH₄)₂SO₄1.0g, 1000mL of water, and neutral pH. 100mL of the crude oil was taken and distributed into 250mL glass bottles with a narrow mouth, 2.0g of the crude oil was added to each bottle, and the mixture was sterilized at 121 ℃ for 30 min.

Preparing a bacterial seed solution: inoculating 1 ring of thallus from the bacterial slant with an inoculating loop, inoculating into 100mL sterilized beef extract peptone liquid medium, and shake culturing at 37 deg.C and 150r/min for 3 d.

Preparing bacterial fermentation liquor: adding 150mL beef extract peptone liquid culture medium into a 600mL tissue culture bottle, sterilizing at 121 ℃ for 30min, cooling, inoculating 5mL bacterial seed solution, shaking and culturing at 37 ℃ and 150r/min for 5d, and storing at 4 ℃ for later use.

NDJ-79 rotational viscometer: shanghai Pinxuan scientific and technological instruments, Inc.

Gas chromatograph: model number was Trace GC Ultra, manufactured by Thermo Finnigan.

Asphalt: the petroleum asphalt is heavy rubber asphalt produced by Shaanxi Baoli asphalt Co., Ltd, and has penetration degree of 91(0.1mm), softening point of 46 ℃, ductility of 45cm at 10 ℃ and ductility of 102cm at 15 ℃.

Crude oil: 6 groups of well oil samples with saturated hydrocarbon content of 612.6g/kg, aromatic hydrocarbon content of 83.6g/kg, colloid of 51.6g/kg, asphalt of 54.3g/kg and other components of 69.1g/kg are prolonged in northern Shaanxi province of China. Viscosity at 35 ℃ of 76.5 mPas.

Example 1

Screening and identification of strains

1. The test method comprises the following steps:

1.1 oil-increasing bacteria separation and purification

Weighing 10.0g of wellhead greasy dirt soil, adding into 90mL of sterile water, and shaking by hand for 15min to obtain 10^-1Standing the soil suspension with the concentration for 30s, sucking 1mL of the soil suspension, adding the soil suspension into a 9mL sterile water pipe, and continuously diluting the soil suspension to 10^-3The spreading method comprises inoculating 3 concentrations of bacterial suspension onto crude oil filter paper of separation medium. Culturing at 37 deg.C for 7 days, selecting single bacterial colony with rapid growth and large diameter, and purifying by dilution plate smearing method to obtain bacteria capable of using crude oil as sole carbon source from crude oil contaminated soil. Shaking crude oil, sucking 0.1mL of crude oil, directly and uniformly coating on a crude oil filter paper of a separation culture medium, and culturing and purifying by the same method to obtain bacteria which can use the crude oil as a unique carbon source from the crude oil. The obtained strain was stored on a beef extract peptone slant.

1.2 preliminary screening and rescreening of the Strain

Primary screening: and (3) inoculating the separated and purified strain into a primary screening culture medium, culturing for 7d at 37 ℃, and observing and recording the growth condition of the strain on the primary screening culture medium at 3-7 d.

Re-screening: and selecting strains which grow well on the primary screening culture medium, inoculating the strains into a secondary screening culture medium, carrying out shake culture at 37 ℃ and 120r/min for 10d, observing the adhesiveness of the crude oil on the bottle wall, and measuring the emulsification time of the crude oil in the culture bottle, the diameter of an oil discharge ring of a culture solution water phase and the pH value. The Relative increase Rate (Δ R%) of each measurement parameter compared to the reference is calculated by the formula (1).

In formula (1): ws and Wck are the emulsification time, drain diameter and pH for treatment and control, respectively.

And (4) integrating the primary screening result and the secondary screening result, and selecting strains which grow well on the primary screening culture medium and the secondary screening culture medium, have long emulsification time and are less in crude oil adhesion on the bottle wall for subsequent tests.

1.3 identification of the Strain

Morphological characteristics: and (3) colony morphology, namely obtaining a single colony by a dilution plate smearing method, and observing the characteristics of colony morphology, size, edge condition, swelling degree, surface gloss, viscosity, colony color and the like. Cell morphology: and (5) observing by a scanning electron microscope. After the sequence is obtained by analyzing the 16S rDNA sequence, similarity search is carried out in NCBI database through Blast program, and a phylogenetic tree is constructed by adopting a Neighbor-join method in Mega 5.0 software.

2. And (3) test results:

32 bacteria are separated from the lengthened oil field crude oil and the oil stain soil, and 1 of the bacteria is the bacteria X9 separated from the crude oil, which grows well in a culture medium containing the crude oil and has obvious influence on the physical and chemical properties of the crude oil. The colony morphology and cell morphology are shown in FIG. 1-FIG. 3, the diameter of X9 colony is 1.8-3.1 mm, the colony is pink, opaque, round, neat in edge, moist and smooth in surface, slightly convex, the cell length is 1.0-2.0 μm, and the cell width is 1.8-3.1 μm

0.1-0.2 μm. The 16S rDNA sequence is as follows:

GTCGAACGGTAAGGCCCTTTCGGGGGTACACGAGTGGCGAACGGGTGAGTAACACGTGGGTAATCTGCCCTGCACTTCGGGATAAGCCTGGGAAACCGGGTCTAATACCGGATATGAGCTCCTGCCGCATGGTGGGGGTTGGAAAGTTTTTCGGTGCAGGATGAGTCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGG GGGATGACGGTCTTCGGATTGTAAACTCCTTTCAGTAGGGACGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCACCGGCCAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTGTCCGGAATTACTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCACGTCGTCTGTGAAATCCTCCAGCTCAACTGGGGGCGTGCAGGCGATACGGGCAGACTTGAGTACTACAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGTAGTAACTGACGCTGAGGAGCGAAAGCATGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGGTGGGCGCTAGGTGTGGGGTCCTTCCACGGATTCCGTGCCGTAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTAGGCTTGACATATACAGGACGACGGCAGAGATGTCGTTTCCCTTGTGGCTTGTATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTGTCTCATGTTGCCAGCACGTTATGGTGGGGACTCGTGAGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCTAGGGCTTCACACATGCTACAATGGCTAGTACAGAGGGCTGCGAGACCGCGAGGTGGAGCGAATCCCTTAAAGCTAGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCTTGTGGAGGGAGCCGTCGA

x9 was identified as Dietzia zerIDiphylli, GenBank accession No. KT 189158.

Example 2

Application of strain

1. Test method

1.1 Effect on crude oil

Adding 18.00g of crude oil into a 500mL triangular flask, adding 200mL beef extract peptone liquid culture medium into each flask, sterilizing at 121 ℃ for 30min, cooling, inoculating 100mL bacterial seed solution, and adding 300mL pure water instead of fermentation liquid as a control. The mixture was allowed to stand at 37 ℃ for 5d while shaking 1 time every 4 hours. And (3) after the treatment is finished, placing the culture bottle in a refrigerator at 4 ℃ for standing, collecting crude oil and a fermentation liquor water phase after the crude oil is solidified, wherein the crude oil is used for measuring the physicochemical property of the crude oil treated by bacteria.

1.2 oil displacing characteristic

pH of the fermentation liquor: the pH value of the water phase of the fermentation liquor is measured by a thunder-magnetic PHS-3D type acidimeter.

Diameter of the oil discharge ring: the diameter of an oil discharge ring of a fermentation liquid water phase is measured by adopting an oil discharge ring method according to the size of the oil discharge ring of liquid culture liquid water in a culture bottle relative to liquid paraffin. The larger the measured value, the stronger the surface activity of the aqueous phase of the fermentation broth, the more surface active substances produced by the growth and metabolism of the bacteria, or the stronger the surface activity.

Adhesion: the attachment condition on the wall of the liquid culture bottle is determined by observing the amount of crude oil attached to the wall of the liquid culture bottle in the bacterial re-screening process. The less crude oil adheres, the cleaner the bottle wall, the greater the reducing effect of the bacterial fermentation broth on the adhesion of the crude oil, and the greater the potential of the fermentation broth for improving the recovery ratio when used for crude oil production. ② the attachment condition of crude oil filter paper, cutting the rapid qualitative filter paper into 4.5cm × 4.5cm filter paper sheets, and measuring the desorption of the fermentation liquor to the filter paper crude oil according to the method in the literature "Bacterial degradation of yield oil soluble forms of bacteria strain isolated from oil-contaminated soil to ceramics microbial enhanced oil recovery application". And recovering the liquid phase after the crude oil filter paper is taken out, and measuring the pH value of the water phase of the control and bacteria treated fermentation liquor and the diameter of the oil discharge ring. The ratio of the amount of crude Oil desorbed from the filter paper to the initial amount of crude Oil adsorbed on the filter paper was defined as the desorption rate (ORE%) of crude Oil adsorbed on the filter paper, and was calculated by equation (2).

In the formula: m is₀、m₁And m₂Respectively indicating the mass of the filter paper sheet, the total mass of the filter paper and the crude oil and the total mass of the residual crude oil and the filter paper after reaction.

Emulsibility: and the crude oil emulsification time refers to the time required for fully shaking up the oil-water mixed liquid culture in the culture bottle during bacterial re-screening, standing on an experiment table and starting timing until the separation of oil and water layers and clear boundary. This value reflects the degree of emulsification of the bacterial culture to the crude oil and its stability.

Viscosity: the influence of the fermentation broth on the viscosity of crude oil was determined using bacterially treated crude oil as the material according to the method described in the literature "Bacterial degradation of microbial oil from oil-contaminated soil recovery application". The viscidity reduction rate (VRR%) of the flooding bacteria was calculated according to equation (3).

In the formula: v_sAnd V_ckViscosity measurements for treatment and control, respectively.

All the above measurements were repeated 3 times.

1.3 treatment of the crude oil with the bacterial fermentation broth for the contents of saturated hydrocarbons, aromatic hydrocarbons, gums and asphaltenes

And (3) asphalt determination: dissolving 2.000g of the crude oil treated by the bacteria in 35mL of normal hexane, standing and settling for 24h, centrifuging at 3500r/min for 5min, and respectively collecting precipitate and an organic liquid phase. And (5) drying and weighing the precipitate in a dryer to obtain the quality of the asphaltene. The supernatant was subjected to alumina column chromatography to determine the crude oil composition.

And (3) measuring the mass of the unknown components: and (3) recovering the alumina, the absorbent cotton and the ammonium sulfate in the chromatographic column after the column chromatography is finished, drying and weighing, and calculating the difference value between the components such as the alumina and the like and the initial mass after the chromatography is finished, so as to obtain the mass of the unknown component remained in the alumina in the elution process. And (3) calculating the content P of each component in the total mass of the crude oil according to the formula (4), and calculating the relative increase rate of each component compared with the control according to the formula (1).

In the formula: w₂And W₁Respectively representing the sum of the empty bottle of a certain component receiving bottle and the mass of the component and the mass of the empty bottle, W_CKIs the total mass of the crude oil.

1.4 degradation of pure asphalt by bacterial fermentation broth

And (3) degradation: 3.000g of pure pitch was weighed accurately and dissolved in 30mL of carbon tetrachloride to prepare a uniform pitch solution. Adding 5 drops of the draining solution into a weighed glass slide (with the mass of m)₀) On the upper part, carefully rotating the glass slide to pave the asphalt solution, and after the carbon tetrachloride is naturally volatilized, the pure asphalt is uniformly attached to the glass slide, wherein the mass of the asphalt glass slide is m₁. Sterilizing the asphalt glass slide for 30min under ultraviolet irradiation, horizontally placing the asphalt glass slide in a sterilized 500mL glass bottle containing 100mL beef extract peptone liquid culture medium, cooling, inoculating 10mL bacterial seed solution, culturing at 37 ℃ for 35d, and gently shaking the tissue culture bottle 4 times every day. After the culture is finished, taking out the asphalt glass slide, washing the asphalt glass slide for 3 times by using distilled water, naturally drying the asphalt glass slide, and weighing the mass m of the asphalt glass slide₂(ii) a Adding 100mL of carbon tetrachloride into the culture solution, shaking thoroughly to dissolve the asphalt entering the culture solution in the organic phase during the action process, recovering the organic phase, evaporating to dryness, and weighing (m)₃) The degradation amount of the bacteria fermentation liquor to the pure asphalt ism₁+m₃-m₂. Each treatment was repeated 3 times. The ratio of the amount of degradation of pitch by the fermentation broth to the initial pitch attachment mass was defined as the pitch degradation rate (ADE%), and calculated according to formula (5). And (3) calculating the increase rate of the bacterial fermentation liquor treatment compared with the control according to the formula (1).

Group composition: the asphalt (mass m) on the surface of the asphalt glass slide after the culture is finished₂-m₀) Dissolving the asphalt with 30mL of normal hexane, and measuring the composition of the asphalt group treated by the bacterial fermentation liquid.

Pitch micro morphology: the pictures of the asphalt glass slide after the bacteria action are taken when the asphalt glass slide is magnified 2 times (reflected light), 20 times (transmitted light) and 40 times (transmitted light), and the change of the micro-morphology of the asphalt attached to the surface of the glass slide is observed.

1.5 Displacement of crude oil by bacterial fermentation broth

1.5.1 oil-displacing material and device

Simulating oil displacement: the oil sample is collected from extended oil fields in northern Shaanxi of China, the saturated hydrocarbon content of the oil sample is 684.50g/kg, the aromatic hydrocarbon content is 108.33g/kg, the colloid content is 39.83g/kg, the asphaltene content is 39.50g/kg, and the unknown component content is 77.00 g/kg.

Bacterial Dietzia cercidiphylli X9 Broth (Fermentation Broth, FB): adding 150mL of bacterial culture medium for displacement of reservoir oil into a 600mL tissue culture bottle, sterilizing at 121 ℃ for 30min, cooling, inoculating 5mL of bacterial Dietziacercindiphylli X9 seed solution (inoculating 1-ring thallus is selected from a bacterial inclined plane by an inoculating ring and inoculated into 100mL of sterilized beef extract peptone liquid culture medium, performing shake culture at 37 ℃ and 150r/min for 3d by a shaking table), performing shake culture at 37 ℃ and 150r/min for 5d by a shaking table, and storing at 4 ℃ for later use.

Simulating an oil displacement device: the oil displacement device comprises an oil displacement device basic component, an oil displacement tube structure and a gas production device, and specifically comprises the following components: pressure gauge a₁And a compressed air inlet b₁And a compressed air outlet c₁And a simulation oil drive pipe d₁Upper inlet and outlet valve a₂Lower inlet/outlet valve e₂And a displacement fluid storage tubeb₂Quick-mounting clamp c₂Oil sand pipe d₂Stainless steel filter plate g₂Cotton filter cloth h₂Sealing washer f₂Sealing glass rod a of gas production pipe₃Gas production syringe needle b₃And a conduit c connected with an outlet at the upper end of the oil displacement pipe₃The components are shown in detail in fig. 6.

1.5.2 preparation of oil displacement pipe for oil displacement simulation

Filling material for the oil sand pipe: fine sand with particle size of 0.15-0.25mm (60-100 mesh), wherein the content of quartz is 78%, feldspar is 6%, and heavy mineral is 16%. Soaking fine sand in 2mol/L diluted hydrochloric acid solution for 12 hr, and washing with tap water to remove CaCO₃The acid soluble salt and the water soluble salt are subjected to iso-dissolving, and then the residual mineral ions are removed by using pure water; drying at 80 deg.C, removing iron filings from the sand grains with magnet, sealing and bagging. Measuring the true volume by a drainage method, and calculating the true density rho to be 2.56g/cm³。

Preparing an oil sand pipe and assembling an oil displacement pipe: laying filter cloth and a stainless steel filter plate at the lower end of the air-oil sand pipe, adding a silica gel gasket, accurately butting with a lower-end valve, combining the air-oil sand pipe and the lower-end valve by using a quick-mounting clamp, and vertically placing; 300.0g of fine sand is uniformly filled into the oil sand pipe from the upper end of the empty oil sand pipe in batches, and a flat-head round wood rod with the diameter of 24mm is tamped after each filling. After all the fine sand is filled, uniformly knocking the outer wall of the oil sand pipe for 5min by using a wood bar to surround the oil sand pipe from bottom to top, and ensuring that the fine sand in the pipe reaches a compact state; and adding filter cloth, a stainless steel filter plate and a silica gel gasket at the upper end of the oil sand pipe filled with fine sand, and connecting the oil sand pipe with a displacement fluid storage pipe and an upper end valve by using a fast-assembling clamp to assemble a complete simulation oil displacement pipe. In the prepared oil sand pipe, the sand core column is 290mm high, the diameter is 29mm, and the volume weight is 1.57g/cm³The porosity of the sand core column was 38.8% as calculated according to equation (6). The sand core Pore Volume (PV) was 74.3cm³。

Filling crude oil: adding 100mL of crude oil into a 200mL beaker, melting in a water bath at 60 ℃, wiping the outer wall of the beaker, and weighing the beakerInitial total mass of cup and crude oil (m)₁) (ii) a Closing a switch at the lower end of the oil displacement pipe, and filling about 100ml of crude oil into a displacement fluid storage pipe; after a gasket is added, a displacement fluid storage pipe is connected with an upper end valve and a compressed air conduit by a quick-mounting clamp; placing the beaker at the outlet at the lower end of the oil sand pipe, opening an upper switch and a lower switch of the oil displacement pipe, introducing 0.1MPa compressed air from the upper end of the displacement fluid storage pipe, pressing crude oil in the displacement fluid storage pipe into the oil sand pipe, and enabling the crude oil to enter the pores of the sand core and be attached to the surface of a sand carrier forming the porous body; and continuously introducing compressed air to enable the redundant crude oil in the oil sand pipe to flow into the receiving beaker at the lower end until no oil drops drop at the outlet, and continuously displacing for 30min by using the compressed air to ensure that the free crude oil in the pores of the sand core column is driven out by the air as much as possible. Closing an upper switch and a lower switch of the oil driving pipe, and weighing the beaker and the total mass (m) of the crude oil in the beaker at the moment₂)，m₁And m₂The difference is the crude oil quality adsorbed by the sand core porous body in the sand tube. And (3) placing the oil displacement pipe in an incubator at 28 ℃ for aging for 24h, so that the crude oil in the sand core porous body of the oil sand pipe is further uniformly distributed on the surface of the sand carrier.

Loosely bound crude oil drive-off: taking out the oil drive pipe after the heat preservation and aging are finished, adding 100mL pure water with the temperature of 45 ℃ into the displacement fluid storage pipe, and quantifying (m)₃) 250mL triangular flask C₁And placing the oil displacement pipe at an outlet at the lower end of the sand core, opening an upper switch and a lower switch of the oil displacement pipe, continuously introducing 0.1MPa of compressed air from the upper end of the displacement fluid storage pipe, closing the air compressor when no water drops flow out from the outlet at the lower end of the oil displacement pipe, and taking the crude oil flowing out along with pure water as loose combined crude oil which can be driven out by water in the pores of the sand core porous body. C is to be₁Cooling in a refrigerator at 4 deg.C, and carefully pouring out the triangular flask C after crude oil is solidified₁Inner pure water, attaching crude oil to the triangular flask C₁On the bottle wall, the triangular bottle C is naturally dried₁Weighing C₁Mass (m)₄)，m₄And m₃The difference is the quality of the water flooding loose bound crude oil. (m)₂-m₁)-(m₄-m₃) Is the initial oil content of the oil sand pipe.

1.5.3 crude oil Displacement

And (3) displacement process: the displacement was carried out for 2 batches, each batch was cultured for 7d at a temperature of 40 ℃. Wherein A, CK and 2 continuous batches are all water flooding. B, FB displacement of X9 fermentation liquor. The displacement method comprises the following steps:

FB displacement: 100mL FB was added to the displacement fluid reservoir of the displacement tubing and the amount (m) determined₅) 250mL triangular flask C₂And (3) placing the reactor below an outlet at the lower end of the sand core, opening a switch at the upper end and the lower end of the oil displacement pipe, slowly introducing 0.1MPa compressed air from the upper end of the oil displacement pipe, closing an air compressor when the fermentation solution flows out of about 20mL drop by drop, returning the FB flowing out to the displacement solution storage pipe again, and repeating the same steps for 3 times to ensure that the FB is filled in the pores of the sand core porous body. And filling the displacement fluid storage pipe with the displacement fluid, closing switches at the upper end and the lower end of the displacement pipe after air is exhausted, and finishing injection. Placing the FB-filled oil displacement pipe in an incubator at 40 ℃ for culturing for 7 d.

Water flooding (CK): FB was replaced with 100mL of pure water, and a total of 2 water displacements were carried out in the same manner as FB.

1.5.4 crude oil driven out and quality determination:

a triangular flask C₂Placing the mixture at the lower end of the oil sand pipe, and continuously introducing compressed air until no fermentation liquor flows out from an outlet at the lower end of the oil sand pipe. Taking another quantified amount (m)₆) 250mL triangular flask C₃Placing the displacement fluid in the lower end of the oil sand pipe, adding 100mL of pure water at 45 ℃ into the displacement fluid storage pipe according to the same step, continuously introducing 0.1MPa of compressed air from the upper end of the displacement fluid storage pipe to drive out residual fermentation liquor in the oil sand pipe, and repeating the water drive process twice. C is to be₂、C₃Cooling in a refrigerator at 4 deg.C, and carefully pouring out the water phase from the triangular flask after the crude oil is solidified. Natural air drying C₂、C₃Attaching the crude oil to the wall of the triangular flask, and weighing C₂Mass (m)₇)，C₃Mass (m)₈). Mass m₈+m₇-m₆-m₅Namely the quality of the adsorption crude oil expelled by the fermentation liquor. The oil displacement Rate ODR% (ODR%) is calculated according to equation (7), and the treatment increase Rate Δ CK% compared with the water displacement treatment is calculated according to equation (8):

in formula (8): m_TAnd M_CKThe values of the parameters for the treatment and the water flooding treatment are indicated separately.

All data were subjected to correlation analysis and significance of differences test using SAS 9.2(SAS Institute Inc, Cary, NC, USA).

2 results and analysis

2.1 Effect of bacterial fermentation broths on the chemical composition and micro-morphology of pure Pitch

As shown in Table 1, after the bacterial fermentation broth treatment, the degradation rate of pure asphalt on the asphalt slide glass by Dietzia cercidiphylli X9 is 9.88 percent, which is 43.0 times of that of the control, and the difference with the control reaches a significant level (P < 0.05). The saturated hydrocarbon in the pure asphalt is increased by 17.1 percent (P <0.05) compared with the control by the treatment of the fermentation liquor of the bacterium Dietzia cercidiphylli X9; the contents of aromatic hydrocarbon, colloid and unknown components are respectively reduced by 20.7%, 27.2% and 40.3% compared with the control (P is less than 0.05).

TABLE 1 degradation rate and family composition of pure asphalt after bacterial fermentation broth treatment

As can be seen in FIG. 4, the micro-morphology of the slide-attached bitumen changed significantly after the bacterial X9 broth treatment. When the glass slide is magnified by 2 times, pure asphalt attached to the surface of the control glass slide is in uniform opaque black, and more transparent spots appear on the surface of the asphalt attached to the X9-treated glass slide. At 20X magnification, the control had only a few small plaques, whereas the X9-treated slides attached plaques much larger in area than the control. At 40 times magnification, the control slide was thin and uniform with bitumen adhered, and the X9 slide was in a somewhat aggregated, elevated state.

2.2 Effect of bacterial fermentation broths on crude oil family composition

As shown in Table 2, the content of saturated hydrocarbon and aromatic hydrocarbon in the crude oil is increased compared with the control and reaches a significant level (P is less than 0.05) after the treatment of the bacterial X9 fermentation liquor; the content of colloid and asphalt is respectively reduced by 44.0 percent and 70.5 percent compared with the control, the difference with the control reaches significant level (P is less than 0.05), the content of unknown components is reduced by 9.8 percent compared with the control, and the content does not reach significant level (P is more than 0.05).

TABLE 2 crude oil family composition (g/kg) after bacterial X9 fermentation broth treatment

Components	Control CK	X9	X9-CK	⊿R％
					1. Asphalt	54.3±3.3a	16.0±2.1b	-39.3	-70.5
2. Glue	51.6±2.6a	28.9±3.0b	-22.7	-44.0
					3. Unknown component	69.1±1.6a	62.3±4.0a	-6.8	-9.8
1-3 components in total	175.0	107.2	-67.8
					4. Saturated hydrocarbons	612.6±5.1b	632.9±3.7a	+20.3	+3.3
5. Aromatic hydrocarbons	83.6±2.3b	112.7±2.3a	+29.1	+34.8
					4-5 components in total	696.2	745.6	+49.4
1-5 components in total	871.2	852.8	-18.4	-2.1

2.3 oil displacing characteristic of bacterial fermentation liquor

2.3.1 surface Activity and emulsifiability: as seen from Table 3, in the liquid culture system using crude oil as a sole carbon source, bacterium X9 improved the emulsifiability of crude oil in the oil-water coexisting system. Wherein, the stabilization time of the emulsion layer treated by the X9 is about 6.6 times of that of the control, and the adhesion of crude oil on the bottle wall can be effectively reduced. The diameter of the oil drain circle of the X9 reaches 16.3cm, which is about 20 times of that of the control, namely, the bacterial strain synthesizes more surface active substances or has stronger activity. When the crude oil is used as the sole carbon source, the pH of the liquid phase of the culture solution of X9 is 6.81, and is not obviously different from that of a control (P is more than 0.05).

TABLE 3 diameter of oil discharge ring of bacterium X9 fermentation liquid and crude oil adhesion on the wall of culture bottle

Note: the crude oil deposits of "+ + + +", "+" and "-" respectively indicate that the bottle wall deposits were large, small, light and almost free of wall sticking.

2.3.2 adhesion: the liquid culture bottle wall treated by the inoculated bacteria X9 is clean, the crude oil is less attached, and the system formed by the crude oil and the water phase has high emulsifying degree. The bacterium X9 produced foam when grown in beef extract peptone broth, indicating that it was capable of forming surface active substances.

2.3.3 desorption of the bacteria X9 fermentation broth on crude oil adsorbed on filter paper: as shown in Table 4, the desorption rate of the crude oil adsorbed on the X9 filter paper after the treatment of the bacterial fermentation liquid is 84.7% which is 2.9 times of that of the control, and the difference with the control reaches a significant level (P < 0.05). During the action of the bacterial fermentation liquor on the filter paper for adsorbing the crude oil, the pH value of the bacterial fermentation liquor is remarkably reduced (P is less than 0.05). As shown in FIG. 5, the residual crude oil amount on the filter paper after the filter paper adsorbing crude oil is treated by the bacterial fermentation liquid is greatly reduced, and the difference from the control CK is obvious.

TABLE 4 crude oil desorption rate of filter paper treated with bacterial fermentation broth

2.3.4 viscosity and oil drainage Activity: as shown in Table 5, the crude oil viscosity at 35 ℃ is remarkably reduced (P is less than 0.05) compared with a control by the action of the bacterial X9 fermentation liquor, and the reduction amplitude is 42.5%; the diameter of the oil discharge ring is 34.0 times of that of the control; after the culture, the pH value of the fermentation liquor is reduced by 1.1 unit, and the difference with the control is obvious (P < 0.05).

TABLE 5 viscosity of crude oil treated with bacterial fermentation broth, desorption rate of crude oil from filter paper, and pH change before and after fermentation broth reaction

Note: the difference is marked in the table by different lower case letters after the same column of data (P < 0.05).

2.4 oil-displacing effect

As shown in Table 7, the total oil displacement rate and the total oil displacement of the X9 fermentation liquor are both significantly higher than those of a control water flooding (P is more than 0.05). In the 2-batch displacement process, the oil displacement rate and the oil displacement of the fermentation liquor treatment are higher than those of a control water flooding, wherein the difference of the 2 nd batch reaches a significant level (P is less than 0.05).

Table 62 displacement amounts and rates

The above examples show that Dietzia cercidiphylli X9 has a strong degradation effect on both bitumen in crude oil and pure bitumen. After the treatment of the bacteria X9, the degradation rate of asphalt in crude oil is 70.5% (P <0.05), and the degradation rate of pure asphalt is 9.9% (P < 0.05). After treatment with the test bacteria X9, the content of saturated hydrocarbons in the pure asphalt increases, and the content of aromatic hydrocarbons, colloids and unknown components decreases. According to the theory of colloid structure, as the content of saturated hydrocarbon and aromatic hydrocarbon of the dispersion medium is reduced, the content of colloid and dispersed phase asphalt of the protective medium is increased, and the asphalt is converted from sol → gel. The research result shows that the thin layer asphalt which is uniformly distributed on the surface of the glass slide originally disappears, then the phenomenon of alternative distribution of an asphalt accumulation area and an asphalt-free transparent area occurs, the phenomenon that the asphalt is changed from gel → sol is proved to occur, and the pure asphalt adhered to the surface of the glass slide is converted into a non-uniform accumulation bump state from a uniform thin layer due to the increase of the fluidity, so that the opposite process occurs when the X9 is used for treating the pure asphalt: the saturated hydrocarbon content of the dispersion medium is increased, and the micro-morphology of the asphalt is consistent with the measurement result of the group composition. In addition, the degradation rate of the test bacteria X9 on the asphalt in the crude oil is far higher than that of the pure asphalt, which indicates that the existence of other light components in the crude oil is helpful for improving the asphalt degradation rate, possibly related to the solubilization of the asphalt by the light components, and in addition, the existence of the light components can provide carbon sources for the growth of the bacteria, promote the metabolism of the bacteria to generate surface active substances, and the existence of the surface active substances promotes the degradation of heavy components in the crude oil by the bacteria. The degradation effect of the test bacterium X9 on asphalt shows that the bacterium has great application potential in the microbial oil increment of the heavy oil reservoir with high asphalt content.

The colloid exists in the crude oil in a stable colloid dispersion state, a complex is easily formed between colloid molecules or between the colloid molecules and asphalt molecules, and the complex is aggregated into a macromolecular aggregate in the stable colloid dispersion state formed by fused ring carboxylic acid and is difficult to degrade. The research finds that the Dietzia cercidiphylli X9 to be tested also has strong degradation effect on the colloid and the unknown high-molecular components in the crude oil and the pure asphalt, and is not reported in the previous research. The colloidal content of crude oil was reduced by 44.0% after treatment with Dietziacercidiphylli X9.

The test Dietzia cercidiphylli X9 has strong surface active substance synthesis ability and acid production ability. In a liquid culture medium with crude oil as a sole carbon source, the bacteria can synthesize surface active substances and produce acid. After the filter paper containing the crude oil is treated by the fermentation liquor of Dietziacercidiphylli X9 to be tested, the filter paper can adsorb and desorb the crude oil, and the surface active substances generated by the metabolism of thalli can reduce the oil-water interfacial tension and greatly reduce the adhesion capability of the crude oil. The bacteria metabolize to produce acid when using the crude oil on the filter paper, and the generated acidic substances can improve the interfacial tension between oil and water, so that the flowability of the crude oil is changed, and the desorption rate is increased.

The total oil displacement amount and the oil displacement rate of the continuous displacement of the crude oil by the Dietzia cercidiphylli X9 fermentation liquor are higher than those of a control water drive, which indicates that the X9 treatment has good crude oil displacement capacity. The oil displacement rates of the 1 st and 2 nd batches of the Dietziacercidiphyll X9 fermentation liquid are respectively 2.8 and 3.2 times of that of the control water flooding.

In conclusion, the tested Dietzia cercidiphylli X9 has stronger degradation capability on asphalt, the process of degrading macromolecular components such as asphalt in the crude oil into micromolecular components changes the physicochemical properties of the crude oil, reduces the adhesiveness and viscosity of the crude oil, and increases the content of light components such as saturated hydrocarbon in the crude oil; the surface active substance generated by the tested bacteria reduces the tension of an oil-water interface, changes the adhesive capacity and the fluidity of the crude oil, greatly improves the oil displacement rate, and has important application value for MEOR and the improvement of the yield of light oil in one-time processing of the crude oil.

Although the present invention has been described in detail in this specification with reference to specific embodiments and illustrative embodiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto based on the present invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

SEQUENCE LISTING

<110> university of agricultural and forestry science and technology in northwest of Shaanxi Boqin bioengineering GmbH

<120> Nectria arborescens and application thereof

<130>2017

<160>1

<170>PatentIn version 3.5

<210>1

<211>1383

<212>DNA

<213>Dietzia cercidiphylli X9

<400>1

gtcgaacggt aaggcccttt cgggggtaca cgagtggcga acgggtgagt aacacgtggg 60

taatctgccc tgcacttcgg gataagcctg ggaaaccggg tctaataccg gatatgagct 120

cctgccgcat ggtgggggtt ggaaagtttt tcggtgcagg atgagtccgc ggcctatcag 180

cttgttggtg gggtaatggc ctaccaaggc gacgacgggt agccggcctg agagggtgat 240

cggccacact gggactgaga cacggcccag actcctacgg gaggcagcag tggggaatat 300

tgcacaatgg gcgaaagcct gatgcagcga cgccgcgtgg gggatgacgg tcttcggatt 360

gtaaactcct ttcagtaggg acgaagcgaa agtgacggta cctgcagaag aagcaccggc 420

caactacgtg ccagcagccg cggtaatacg tagggtgcaa gcgttgtccg gaattactgg 480

gcgtaaagag ctcgtaggcg gtttgtcacg tcgtctgtga aatcctccag ctcaactggg 540

ggcgtgcagg cgatacgggc agacttgagt actacagggg agactggaat tcctggtgta 600

gcggtgaaat gcgcagatat caggaggaac accggtggcg aaggcgggtc tctgggtagt 660

aactgacgct gaggagcgaa agcatgggga gcaaacagga ttagataccc tggtagtcca 720

tgccgtaaac ggtgggcgct aggtgtgggg tccttccacg gattccgtgc cgtagctaac 780

gcattaagcg ccccgcctgg ggagtacggc cgcaaggcta aaactcaaag gaattgacgg 840

gggcccgcac aagcggcgga gcatgtggat taattcgatg caacgcgaag aaccttacct 900

aggcttgaca tatacaggac gacggcagag atgtcgtttc ccttgtggct tgtatacagg 960

tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg 1020

caacccctgt ctcatgttgc cagcacgtta tggtggggac tcgtgagaga ctgccggggt 1080

caactcggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgtc tagggcttca 1140

cacatgctac aatggctagt acagagggct gcgagaccgc gaggtggagc gaatccctta 1200

aagctagtct cagttcggat tggggtctgc aactcgaccc catgaagtcg gagtcgctag 1260

taatcgcaga tcagcattgc tgcggtgaat acgttcccgg gccttgtaca caccgcccgt 1320

cacgtcatga aagtcggtaa cacccgaagc cggtggccta acccttgtgg agggagccgt 1380

cga 1383

Claims

1. A strain of Nectria arborescens, specifically Dietzia cereidiphili X9, is preserved in China center for type culture Collection 3 months and 14 days 2016, and has a preservation number of CCTCC NO: m2016120.

2. The application of Nectria androsaceus X9 in asphalt degradation is characterized in that the Nectria androsaceus X9 is specifically Dietziacercidiphylli X9, is preserved in the China center for type culture Collection at 3 months and 14 days in 2016, and has the preservation number of CCTCCM 2016120.

3. The application of Nectria androsaceus X9 in gum degradation is characterized in that the Nectria androsaceus X9 is specifically Dietziacercidiphylli X9, is preserved in the China center for type culture Collection at 3 months and 14 days in 2016, and has the preservation number of CCTCCM 2016120.

4. The application of Nectria androsaceus X9 in aromatic hydrocarbon degradation is characterized in that the Nectria androsaceus X9 is specifically Dietziacercidiphylli X9, is preserved in the China center for type culture Collection at 3 months and 14 days of 2016, and has the preservation number of CCTCCM 2016120.