CN111394271A

CN111394271A - Acinetobacter for producing surfactant and application thereof

Info

Publication number: CN111394271A
Application number: CN201911109720.0A
Authority: CN
Inventors: 章春芳; 黄小敏; 周航海; 李平原; 程萍; 周少雄; 卜魁勇; 赵波; 吴君; 魏静
Original assignee: XINJIANG KELI NEW TECHNOLOGY DEVELOPMENT CO LTD; Zhejiang University ZJU
Current assignee: XINJIANG KELI NEW TECHNOLOGY DEVELOPMENT CO LTD; Zhejiang University ZJU
Priority date: 2019-09-12
Filing date: 2019-11-13
Publication date: 2020-07-10
Anticipated expiration: 2039-11-13
Also published as: CN111394271B

Abstract

The invention relates to the field of microorganisms, and discloses a surfactant-producing acinetobacter and application thereof, wherein the acinetobacter is named as Y2, is preserved in China Center for Type Culture Collection (CCTCC) in 29 months in 2019, has a preservation number of M2019588, and is classified and named as microorganismAcinetobactersp. 16S DNA sequence of the Y2 is shown in SEQ ID NO. 1. the acinetobacter Y2 obtained in the invention has the functions of generating biosurfactant and degrading hydrocarbons, and the produced biosurfactant of the acinetobacter Y2 has high activity and shows strong tolerance in the ranges of-pH (2-12), temperature (4-100 ℃) and salinity (0-100 g/L).

Description

Acinetobacter for producing surfactant and application thereof

Technical Field

The invention relates to the field of microorganisms, in particular to acinetobacter for producing a surfactant and application thereof.

Background

The fracturing flow-back fluid is a product generated in the fracturing construction operation of petroleum drilling and production, and mainly comes from a large amount of well washing wastewater generated by the action of active water well washing before and after construction, a fracturing gel breaking fluid which is returned from a shaft after construction and a residual fracturing base fluid after construction. The fracturing flowback fluid contains impurities such as iron ions, petroleum, soluble solids, suspended matters, stratum infiltration water and the like, and can be recycled or externally discharged after effective treatment.

As can be seen from the above, the fracturing flow-back fluid has the characteristics of high COD, high viscosity, complex components and the like, so that the physical and chemical treatment methods such as coagulation, Fe/C micro-electrolysis, activated carbon adsorption and the like are mainly adopted for treating the fracturing flow-back fluid in the prior art, for example, Chinese patent with application number CN201811569372.0 discloses a fracturing flow-back fluid treatment method which comprises the following steps of 1) recovering the fracturing flow-back fluid into a recovery tank, carrying out physical treatment on the fracturing flow-back fluid recovered from the recovery tank, removing mechanical impurities, suspended solid impurities and oil contamination impurities in the fracturing flow-back fluid, and carrying out water quality detection and analysis on the fracturing flow-back fluid after the physical treatment, 2) adjusting the pH value to be between 6 and 9 by using a pH adjusting agent according to the pH value obtained in the step 1), then carrying out gel breaking treatment on the fracturing flow-back fluid after the pH value is adjusted by using a gel breaker of 0.002-80 mg/L, and carrying out oxidation treatment by using an oxidant of 0.01-200 mg/L after the gel breaking treatment is finished, so as to obtain the fracturing flow-back fluid with the completely reduced viscosity.

However, the physicochemical treatment methods of the above patents and the like have disadvantages that: the method is easy to cause secondary pollution to the environment and has high cost. Biological processes are processes that use naturally occurring organisms to break down organic matter in wastewater into less toxic or non-toxic substances. The biological method for treating the wastewater has the advantages of environmental friendliness, no secondary pollution, low cost, complete degradation and the like. However, there are few reports in the literature that the fracturing flowback fluid can be repaired by a microbial treatment method, and the reason for analyzing the repairing may be that: on one hand, the fracturing flow-back fluid has complex composition and bad environment (salinity, pH and the like) and is not suitable for the survival of a plurality of microorganisms; on the other hand, the pH fracturing flow-back fluid contains a large amount of hydrophobic organic matters, so that the degradation and utilization of microorganisms are limited, the biodegradability is poor, and ideal microorganisms suitable for biochemical treatment of the fracturing flow-back fluid cannot be found.

Disclosure of Invention

In order to solve the above technical problems, the present invention provides a surfactant-producing acinetobacter and its use, wherein the obtained acinetobacter Y2 has both functions of producing biosurfactant and degrading hydrocarbons, and the biosurfactant produced by the acinetobacter Y2 not only has high activity, but also shows strong tolerance in the range of-pH (2-12), temperature (4-100 ℃) and salinity (0-100 g/L).

The specific technical scheme of the invention is as follows:

in a first aspect, the present invention provides a surfactant producing acinetobacter, named as Y2, which has been deposited in the chinese type culture collection center at 29 months 7 and 2019 with a deposition number of CCTCC NO: m2019588, microbial classification named Acinetobacter sp; the 16S DNA sequence of the Y2 is shown in SEQ ID NO. 1.

As described in the background, due to the complex composition of the frac flowback fluid, the inclusion of a large amount of hydrophobic organics limits the degradation and utilization thereof by microorganisms, and is less biodegradable. Therefore, there is no method for treating microorganisms. Therefore, the microbial treatment method is successfully applied to the restoration of the fracturing flow-back fluid, and the surfactant is added to improve the solubility of pollutants in the wastewater in the early stage of the team, so that the biodegradability of the pollutants is improved. However, the synthetic surfactant has poor biodegradability, high low critical micelle concentration and high toxicity, and the activity of the synthetic surfactant is easily influenced by the pH, temperature, salinity and the like of the water environment, so that the synthetic surfactant is limited in practical application.

The acinetobacter Y2 is derived from a Xinjiang Cramalima 18 well area, further experiments show that the surfactant produced by Y2 is lipopeptide, the critical micelle concentration is 187 mg/L, the surface tension of water can be reduced from 72mN/m to 30.21mN/m, and the performance of the surfactant can be kept stable under different ranges of pH, temperature and NaCl concentration, and the application of Y2 to the fracturing flowback fluid for in-situ biological enhanced repair is found to remarkably enhance the microbial activity and promote the degradation of COD (from 6646.7 mg/L to 1546.7 mg/L in 7 days) and normal alkanes (2635.4 mg/L to 159.7 mg/L) and polycyclic aromatic hydrocarbons (918.6 mu g/L to 209.6 mu g/L) in the fracturing flowback fluid.

From the above, it is understood that the acinetobacter Y2 of the present invention can produce a biosurfactant itself, and the biosurfactant produced therefrom has characteristics of better biodegradability, low toxicity, high efficiency, low critical micelle concentration, and the like, as compared with a synthetic surfactant. Meanwhile, the acinetobacter Y2 can also remarkably enhance the activity of microorganisms in the fracturing flow-back fluid and promote the degradation of COD and normal alkane and polycyclic aromatic hydrocarbon therein. In addition, the acinetobacter Y2 is derived from fracturing flowback fluid, belongs to indigenous bacteria, and compared with exogenous bacteria, the indigenous bacteria has better environment adaptability (the fracturing flowback fluid is complex in composition, and the environment (salinity, pH and the like) is severe, so that the fracturing flowback fluid is not suitable for the survival of a plurality of microorganisms), and can better promote the degradation of pollutants. Therefore, compared with other strains, the acinetobacter Y2 has the three characteristics of good biosurfactant performance, excellent organic matter degradation capability and strong adaptability to the fracturing flowback fluid, and can efficiently repair the fracturing flowback fluid under the cooperation of the three characteristics.

The acinetobacter of the present invention also includes a culture of Y2 or a culture after passaging.

In a second aspect, the present invention provides a surfactant-producing acinetobacter mutant, which is obtained by subjecting acinetobacter Y2 to mutagenesis, acclimation, genetic recombination or natural mutation.

In a third aspect, the present invention provides a bacterial culture of an acinetobacter Y2 or Y2 mutant.

In a fourth aspect, the invention provides a fermentation method of acinetobacter Y2, which comprises the following specific fermentation conditions of adopting olive oil as a carbon source, ammonium sulfate as a nitrogen source, fermentation temperature of 28-32 ℃, salinity of 8-12 g/L and pH of 6.8-7.2.

Preferably, the concentration of the olive oil is 0.8-1.2wt%, and the concentration of the ammonium sulfate is 1.5-2.5 g/L.

In a fifth aspect, acinetobacter Y2 or a mutant thereof of the present invention can be used in frac flowback fluid repair.

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

(1) the invention screens a surfactant producing strain Y2 from a Xinjiang Cramar 18 well area, and the surfactant producing strain is identified as the acinetobacter by 16S DNA further experiments show that the surfactant produced by Y2 is lipopeptide, the critical micelle concentration is 187 mg/L, the surface tension of water can be reduced from 72mN/m to 30.21mN/m, and the performance of the surfactant can be kept stable in the ranges of pH (2-12), temperature (4-100 ℃) and salinity (0-100 g/L), and the surfactant producing strain shows strong tolerance.

(2) The biosurfactant is characterized as lipopeptide by T L C, FTIR and GC-MS analysis, and the removal of COD and hydrocarbons (including normal alkanes and PAHs) can be remarkably promoted by adding acinetobacter Y2.

(3) The acinetobacter Y2 is derived from fracturing flowback fluid, belongs to indigenous bacteria, and compared with exogenous bacteria, the indigenous bacteria has better environment adaptability (the fracturing flowback fluid is complex in composition, and the environment (salinity, pH and the like) is severe and is not suitable for the survival of a plurality of microorganisms), and can better promote the degradation of pollutants.

(4) The invention discloses that Y2 is applied to fracturing flowback fluid for in-situ biological enhanced repair, which can remarkably enhance the microbial activity, promote COD (from 6646.7 mg/L to 1546.7 mg/L within 7 days) and degradation of normal alkane (2635.4 mg/L to 159.7 mg/L) and polycyclic aromatic hydrocarbon (918.6 mu g/L to 209.6 mu g/L).

Drawings

FIG. 1 is a graph showing the results of various performance tests of Y2; wherein, the hemolytic cycle experiment (a); oil drain ring experiment (b); droplet collapse experiment (c); index of emulsifiability E₂₄(d) (ii) a Y2 emulsified crude oil test (e) left: control (water), right: experimental group (Y2 broth); phylogenetic tree of strain Y2 (f).

FIG. 2 is a graph showing the results of performance tests on the resulting surfactant of Y2; wherein CMC is measured (a); y2 surfactant stability under varying conditions of pH, temperature and NaCl concentration. E₂₄Is the emulsification index (%); ST represents surface tension (mN/m) (b).

FIG. 3 is a graph showing the detection results of Y2, wherein the T L C, FTIR results and GC-MS analysis results of Y2 biosurfactant are shown in the graph (a), the graph (b) and the graph (C).

Fig. 4 shows the effect of Y2 on the removal of organics in frac flowback fluid. Wherein the COD values and corresponding removal efficiencies (a) of the different treatment groups; concentration of total n-alkanes and n-alkanes of different chain lengths under different treatments (b); concentrations of PAHs and different cyclic PAHs under different treatments (c). Error bars indicate standard deviation. Indicates significant differences between the original samples and other treatments (P < 0.05) or very significant differences (P < 0.001).

FIG. 5 shows the surface tension (a), OD of the different treatment groups₆₀₀And FDA hydrolytic activity (b). Error bars represent standard deviation.

Detailed Description

The present invention will be further described with reference to the following examples.

General examples

The invention provides an acinetobacter, which is named as Y2 and is preserved in China center for type culture Collection in 29 7 months in 2019, wherein the preservation number is CCTCC NO: m2019588, microbial classification named Acinetobacter sp; the 16S DNA sequence of the Y2 is shown in SEQ ID NO. 1.

The present invention provides cultures or subcultures of Y2.

The invention provides a mutant of acinetobacter Y2, which is obtained by carrying out mutagenesis, domestication, gene recombination or natural mutation on acinetobacter Y2.

The present invention provides a cell culture of Acinetobacter Y2 or a mutant thereof.

The invention provides a fermentation method of acinetobacter Y2, which comprises the following fermentation conditions of adopting olive oil as a carbon source, ammonium sulfate as a nitrogen source, wherein the fermentation temperature is 28-32 ℃, the salinity is 8-12 g/L, and the pH is 6.8-7.2. preferably, the concentration of the olive oil is 0.8-1.2wt%, and the concentration of the ammonium sulfate is 1.5-2.5 g/L.

The invention provides application of acinetobacter Y2 or mutants thereof in repairing fracturing flowback fluid.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

Performance testing of Acinetobacter Y2

1. Oil drainage Ring test A clean 10cm dish was filled with 30m L ultrapure water, 100. mu. L light crude oil was added dropwise to the water, the crude oil rapidly diffused on the water surface, after it stabilized, 10. mu. L supernatant broth was slowly added dropwise with a pipette, the diameter of the oil drainage ring was measured, and the result was shown in FIG. 1(b) with the addition of an equal volume of ultrapure water as a blank.

2. Experiment of drop collapse

25 μ L cell-free supernatant was dropped onto the sealing film, followed by observing the shape of the droplet and the spreading of the droplet on the surface of the sealing film, then methylene blue (which has no effect on the shape of the droplet) was added to the droplet for photographing, and the diameter of the droplet was measured with a ruler to drop an equal volume of ultrapure water as a blank control, the results are shown in FIG. 1 (c).

3. Measuring surface tension of fermentation liquor

The surface tension of the fermentation broth was measured at room temperature using a surface tensiometer (BZY-201, Shanghai Fangrui Instrument Co. L td., China).

4. Determination of the emulsification index

Adding 3ml of the centrifuged fermentation liquid and 3ml of mineral oil into a test tube, treating for 10min by using an ultrasonic instrument, completely mixing, standing for 24 hours at room temperature, and determining an emulsification index:

the results are shown in FIG. 1 (d); the Y2 emulsified crude oil test is shown in fig. 1(e), left: control (water), right: experimental group (Y2 broth).

The results of the above tests are shown in table 1:

TABLE 1 comparison of data for Acinetobacter Y2 and other strains

Strain numbering	Diameter of oil discharge ring (mm)	Surface tension value (mN/m)	Emulsification index E₂₄(％)	Diameter of collapsed droplet (mm)
					F1	65±2.3	36.17±0.45	29.68±2.1	4±0.5
F2	52±1.8	40.21±0.57	20.04±2.4	3.5±1
					Y2	100±1.0	26.08±0.38	53.35±0.6	6±1.0
Y3	70±1.2	35.13±0.12	32.12±1.5	5±1.5

Wherein, F1 is acinetobacter junii, F2 is enterobacter carvaceae, and Y3 is pseudomonas aeruginosa. As can be seen from the data in table 1, Y2 of the present invention has significant advantages over other strains in the experiments of oil drain diameter, surface tension value, emulsification index, and droplet collapse.

5. Identification of strains

Based on the above screening, the strain with the highest biosurfactant-producing ability was selected for molecular identification. DNA was extracted using the easy pure bacterial Genomic DNA Kit (Transgene Biotechnology Co., L td., Beijing, China). the obtained DNA was used as template DNA for Polymerase Chain Reaction (PCR). the 16S DNA gene amplicon was sequenced (TSINGKE Biotechnology Co., L td., Handzhou, China) at 27f and 1492 r. the comparison results were obtained by IC L acid Alignment Search Tool (https:// blast. ncbi. n.lm. nih gov.) A phylogenetic tree was constructed using MEGA7.0 software (Pesylvania Stateuniversity, State College, USA, PA) in which the sequence of Acinetobacter Y. 2 is shown in SEQ ID No. 361.

6. Biosurfactant extraction and performance characterization

6.1 extraction of biosurfactants

Strain Y2 was cultured for 5 days in MSM medium containing 1% olive oil as a carbon source. The culture solution was centrifuged at 8000g for 10 minutes to collect the supernatant. The supernatant was extracted three times with ethyl acetate. The extracts were combined and concentrated using a rotary evaporator.

6.2 determination of the Critical Micelle Concentration (CMC)

Preparing the extracted biosurfactant into a series of biosurfactant solutions with different concentrations, and measuring the surface tension of the solutions by using a surface tension meter. The surface tension of the solution decreases with increasing surfactant concentration, and when the surface tension of the solution becomes stable and does not decrease, the corresponding concentration is the critical micelle concentration of the surfactant. As a result, as shown in FIG. 2(a), the CMC of the biosurfactant produced by Y2 was 187.5mN/m, corresponding to a surface tension of 30.2 mN/m.

6.3 biosurfactant stability assay

① stability to temperature surfactant was dissolved in 50ml water to prepare surfactant solutions, which were treated at different temperatures (20, 40, 60, 80, 100 ℃) for 30 minutes, and the surface tension of the solutions after each temperature treatment was measured.

② stability to pH the surfactant solutions were adjusted to different pH's (2-12) with 3M hydrochloric acid and 3M NaOH solutions and the surface tension of the solutions at the different pH's was measured.

③ stability to salinity the surface active agent solutions were tested for surface tension at different salinity by adding different amounts of NaCl to the prepared surfactant solutions to vary the salinity of the solutions from 0 to 100 g/L.

The specific results are shown in FIG. 2(b) according to FIG. 2(b), the extracted biosurfactant shows strong tolerance in the range of pH (2-12), temperature (4-100 ℃) and salinity (0-100 g/L).

6.4 thin-layer chromatography (T L C)

A portion of the biosurfactant was dissolved in methanol and approximately 10. mu. L solution was spotted on a silica gel plate (Marine BiotechCo., Qingdao, China.) A mobile phase separation compound using chloroform/methanol/water (95: 5: 1, v/v/v) Ninhydrin reagent (0.25% Ninhydrin in acetone) was used to detect the peptide brick red spots, the silica gel plate was treated with iodine vapor and the lipids developed yellow spots, as shown in FIG. 3(a) is the T L C pattern for Y2 biosurfactant, which was sprayed with 0.25% Ninhydrin solution onto the A plate to detect the peptide, the red was positive, and the B plate was developed with iodine vapor to detect the lipids, the lemon yellow was positive.

6.5 FTIR

To identify the chemical bond type of biosurfactant, FTIR spectrometer (CCR-1, Thermo-Nicolet, America) was used at 4000cm^-1-400cm^-1Performing Fourier transform infrared spectroscopy within the spectral range of (1). FTIR results are shown in FIG. 3 (b);

6.6 gas chromatography Mass Spectrometry (GC-MS) analysis of fatty acid composition

In order to verify the fatty acid component of the surfactant produced by strain Y2, the surfactant produced by strain Y2 was hydrolyzed and methyl-esterified, then extracted and concentrated with n-hexane, and subjected to GC-MS analysis of the fatty acid component and structure. 10mg of the extracted surfactant was weighed out and added to 5ml of 2M HCl-CH₃In OH (1: 11), an ampoule tube is sealed by filling nitrogen, the reaction is carried out for 4 hours in water bath at 100 ℃, and after the reaction is cooled, 2ml of n-hexane is used for extraction twice. Mixing extractive solutions in centrifuge tube with plugMedium dilution 100 times for GC-MS analysis, the instrument is Shimadzu QP2020 gas chromatograph/mass spectrometer, helium gas as carrier gas, column flow rate 1.5ml/min, injection port temperature 260 ℃, gas interface temperature 260 ℃, column initial temperature 60 ℃, temperature rise rate 5 ℃/min up to 260 ℃, and keeping 10min, mass spectrometry conditions, ion source temperature 200 ℃, scanning range 50-500 amu, sample introduction amount 1 mu L, and split ratio 50: 1, searching the structure ratio GC-MS result of fatty acid methyl ester in the National Institute of Standards and Technology (NIST) mass library database to estimate possible fatty acid composition of the biosurfactant, GC-MS analysis result shown in figure 3(C), and FTIR result show that the biosurfactant produced by Y2 is lipopeptide, after methyl esterification, GC-MS analysis of fatty acid composition hexadecanoic acid and octadecanoic acid

7. Biological repair of fracturing flowback fluid

7.1 Experimental group settings

In order to test whether indigenous biosurfactant-producing bacteria can adapt to the fracturing flow-back fluid and promote the removal of COD, normal paraffin and polycyclic aromatic hydrocarbon, Y2 is added into the fracturing flow-back fluid, and the removal effects of the biosurfactant-producing bacteria on COD, normal paraffin and PAHs are respectively evaluated, for a biodegradation experiment, 100m L fracturing flow-back fluid is added into a 250m L conical flask.

The set groups were set as follows, the control group was supplemented with 1m L sterile ultrapure water, the biostimulation group provided 1m L10 g/L Yeast Extract (YE), and the bioaugmentation group was provided with 1m L mixture (Y2 cells and 10 g/L YE).

The solution pH was adjusted to 7.0-7.5 and all supplements were added daily regularly during the first three days of culture. All experimental groups were incubated at 30 ℃ for 7 days with shaking at 180 rpm.

7.2 COD and Hydrocarbon component analysis

After the end of the culture, the culture was centrifuged at 8000g for 10 minutes to remove the cells, the COD value was determined by using the dichromate method, the residual oil was extracted and analyzed by GC-MS for determining the degradation efficiency of the different treatment groups, briefly, 3m L culture was added to an equal volume of n-hexane to recover the residual oil in the culture, the extraction was repeated three times, the upper organic phases were combined, and the anhydrous phase was usedNa₂SO₄Finally, the n-hexane phase was diluted 10-fold and the fractions of C8-C40 were quantified by GC-MS (QP2020, Shimadzu) equipped with SH Rxi-5Sil MS column (30m × 0.25 μm × 0.25.25 mm, Shimadzu) helium was used as a carrier gas at a flow rate of 1.2m L/min and column oven temperature parameters were set such that the initial temperature was set at 50 ℃, the retention time was 2 minutes, the temperature was 6 ℃/minute, the temperature was raised to 300 ℃, the retention time was 25 minutes, the ion source and interface temperature were set at 230 and 300 ℃ respectively, the collection mode was set as the selected ion monitoring mode, the ions of each fraction corresponded to the retention time of external standards (34 alkanes). furthermore, as described by Sun et al, the remaining PAHs in the culture solution were extracted and analyzed with reference to (EPA) -PAHs specified by the U.S.A.

FIG. 4(a) shows the COD values and corresponding removal efficiencies of different treatment groups, wherein the original sample is 6646.7 mg/L, the control water-adding group is 6446.7 mg/L (removal rate 3%), the biostimulation group is 5246.7 mg/L (removal rate 21.1%), and the bioaugmentation group is 1546.7 mg/L (removal rate 76.7%), and the results show that the strain Y2 generating the biosurfactant can be well adapted to the complex environment in the fracturing flowback fluid and effectively promote the removal of COD.

FIG. 4(b) shows the concentrations of total n-alkanes and different chain length n-alkanes at different treatments the initial total n-alkane (C8-C40) content was 2635.4 mg/L, and C8-C40 contents were 2339.6 mg/L (removal rate 11.2%), 1380.4 mg/L (removal rate 47.6%) and 159.7 mg/L (removal rate 93.9%) in the control water-addition, biostimulation and bioaugmentation treatment groups, respectively.

Figure 4(c) shows the concentrations of PAHs and different ring PAHs under different treatments, error bars indicate standard deviation, indicating significant differences (P < 0.05) or very significant differences (P < 0.001) between the original sample and the other treatments, wherein the original sample was 918.6 μ g/L, the control watered group was 760.6 μ g/L (removal rate 17.2), the biostimulated group was 556.8 μ g/L (removal rate 39.4%), the bioaugmented group was 209.6 (removal rate 77.2%), the results show that the addition of acinetobacter Y2 significantly promoted the degradation of PAHs in frac flowback fluid and the highest content of 3-and 4-ring aromatics in PAHs was effectively degraded (removal rate 79.3%).

7.2 measurement of surface tension of culture solution

To examine whether or not biosurfactants were produced in the culture broth, the surface tension of the culture supernatant was measured as an indirect index using a surface tensiometer (BZY-201, Shanghai Fangrui Instrument Co. L td., China). the results are shown in FIG. 5 (a).

7.3 determination of microbial growth and Activity

The culture medium of 0.5m L was centrifuged to collect the cells, and 7.5m L phosphate buffer (NaCl 8.5 g/L) was added₂PO₄0.1g/L，Na₂HPO₄2.2 g/L, pH 7.6), shaking at 200rpm, 30 ℃ for 15 minutes, then 0.25m L Fluorescein Diacetate (FDA) solution (2 g/L FDA in acetone) was added and the mixture was shaken at 200rpm, 30 ℃ for 2 hours, after the end of the culture the cells were centrifuged off and the absorbance of the supernatant was measured at 490nm, the results are shown in FIG. 5 (b).

The results in fig. 5 show that the addition of acinetobacter Y2 significantly reduced the surface tension of the fermentation broth, indicating that a large amount of biosurfactant was produced and enhanced the growth and activity of microorganisms in the frac flowback fluid.

7.4 data analysis

All data are mean values of 3 replicates. To test for significant differences in COD between the different treatments, a t-test analysis was performed using SPSS19.0 software (IBM corp., USA).

In conclusion, the CMC of the biosurfactant produced by Acinetobacter Y2 was 187.5 mg/L and showed strong tolerance in different pH (2-12), temperature (4-100 ℃) and salinity (0-100 g/L) ranges.

The addition of Acinetobacter Y2 significantly facilitated the removal of COD and hydrocarbons (including normal alkanes and PAHs).

In addition, the biomass and activity of the microorganisms were greatly increased after the addition of Y2 to the frac flowback fluid. The study proves that the microorganisms inoculated with the indigenous surfactant are an effective method for bioremediation of the pressure flow-back fluid.

The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.

Sequence listing

<110> Zhejiang university

<120> acinetobacter producing surfactant and use thereof

<130>2019

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>1445

<212>DNA

<213> Acinetobacter Y2(Acinetobacter sp. Y2)

<400>1

agccggggcg gcagcttaca catgcagtcg agcgggggaa ggtagcttgc tactggacct 60

agcggcggac gggtgagtaa tgcttaggaa tctgcctatt agtgggggac aacattccga 120

aaggaatgct aataccgcat acgtcctacg ggagaaagca ggggaccttc gggccttgcg 180

ctaatagatg agcctaagtc ggattagcta gttggtgggg taaaggccta ccaaggcgac 240

gatctgtagc gggtctgaga ggatgatccg ccacactggg actgagacac ggcccagact 300

cctacgggag gcagcagtgg ggaatattgg acaatggggg gaaccctgat ccagccatgc 360

cgcgtgtgtg aagaaggcct tatggttgta aagcacttta agcgaggagg aggctactag 420

tattaatact actggatagt ggacgttact cgcagaataa gcaccggcta actctgtgcc 480

agcagccgcg gtaatacaga gggtgcgagc gttaatcgga tttactgggc gtaaagcgtg 540

cgtaggcggc catttaagtc aaatgtgaaa tccccgagct taacttggga attgcattcg 600

atactggatg gctagagtat gggagaggat ggtagaattc caggtgtagc ggtgaaatgc 660

gtagagatct ggaggaatac cgatggcgaa ggcagccatc tggcctaata ctgacgctga 720

ggtacgaaag catggggagc aaacaggatt agataccctg gtagtccatg ccgtaaacga 780

tgtctactag ccgttggggc ctttgaggct ttagtggcgc agctaacgcg ataagtagac 840

cgcctgggga gtacggtcgc aagactaaaa ctcaaatgaa ttgacggggg cccgcacaag 900

cggtggagca tgtggtttaa ttcgatgcaa cgcgaagaac cttacctggc cttgacatac 960

tagaaacttt ccagagatgg attggtgcct tcgggaatct agatacaggt gctgcatggc 1020

tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttttc 1080

cttacttgcc agcatttcgg atgggaactt taaggatact gccagtgaca aactggagga 1140

aggcggggac gacgtcaagt catcatggcc cttacggcca gggctacaca cgtgctacaa 1200

tggtcggtac aaagggttgc tacctagcga taggatgcta atctcaaaaa gccgatcgta 1260

gtccggattg gagtctgcaa ctcgactcca tgaagtcgga atcgctagta atcgcggatc 1320

agaatgccgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accatgggag 1380

tttgttgcac cagaagtagg tagtctaacc gcaaggagga cgctaccacg gtgccccatt 1440

ttgca 1445

Claims

1. A surfactant-producing Acinetobacter, characterized in that: said Acinetobacter is named Y2 and has been deposited in 2019 on 7/29 thThe national type culture collection center has a collection number of CCTCC NO: M2019588, and the classification name of the microorganism isAcinetobactersp.; the 16S DNA sequence of the Y2 is shown in SEQ ID NO. 1.

2. The acinetobacter of claim 1, which is a culture of said Y2 or a culture after passaging.

3. A mutant of acinetobacter producing a surfactant, which is obtained by subjecting the acinetobacter of claim 1 or 2 to mutagenesis, acclimation, genetic recombination or natural mutation.

4. A bacterial culture comprising the Acinetobacter according to claim 1 or 2 or the mutant according to claim 3.

5. A fermentation method of Acinetobacter according to claim 1, wherein the fermentation conditions are olive oil as a carbon source, ammonium sulfate as a nitrogen source, the fermentation temperature is 28-32 ℃, the salinity is 8-12 g/L, and the pH is 6.8-7.2.

6. The fermentation method according to claim 5, wherein the concentration of olive oil is 0.8-1.2wt% and the concentration of ammonium sulfate is 1.5-2.5 g/L.

7. Use of the acinetobacter of claim 1 or 2 or the mutant of claim 3 for frac flowback fluid repair.