Method of microbial enhanced oil recovery by changing microbial motility

Abstract

Disclosed herein is a method of microbial enhanced oil recovery by changing microbial motility, including: subjecting a viable Glycocaulis strain and the alkane-degrading Dietzia strain to contact culture to enable the alkane-degrading Dietzia strain to have motility. This application also provides a method for microbial enhanced oil recovery, including: subjecting a viable Glycocaulis strain and the alkane-degrading Dietzia strain to contact culture to enable the alkane-degrading Dietzia strain to have motility; and injecting the contact culture mixture of the viable Glycocaulis strain and the alkane-degrading Dietzia strain to an oil well to perform microbial enhanced oil recovery.

Claims

1. A method of changing a motility of an alkane-degrading Dietzia strain, comprising: subjecting a viable Glycocaulis strain and an alkane-degrading Dietzia strain to contact culture to enable the alkane-degrading Dietzia strain to have motility, wherein the viable Glycocaulis strain is viable Glycocaulis sp. 6B-8.

2. The method of claim 1, wherein the alkane-degrading Dietzia strain is Dietzia sp. DQ12-45-1b.

3. A method for microbial enhanced oil recovery, comprising: subjecting a viable Glycocaulis strain and an alkane-degrading Dietzia strain to contact culture to enable the alkane-degrading Dietzia strain to have motility; and injecting the contact culture mixture of the viable Glycocaulis strain and the alkane-degrading Dietzia strain to an oil well to perform microbial enhanced oil recovery; wherein the viable Glycocaulis strain is viable Glycocaulis sp. 6B-8; and the alkane-degrading Dietzia strain is Dietzia sp. DQ12-45-1b.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A-D illustrates the change of colony radius of Dietzia sp. DQ12-45-1b and Glycocaulis sp. 6B-8 over time. FIG. 1A: Dietzia sp. DQ12-45-1b; FIG. 1B: Glycocaulis sp. 6B-8; FIG. 1C: Dietzia sp. DQ12-45-1b with viable Glycocaulis sp. 6B-8 cells; and FIG. 1D: Dietzia sp. DQ12-45-1b with dead Glycocaulis sp. 6B-8 cells.

(2) FIG. 2 shows the real-time observation results of the contact culture of Dietzia sp. DQ12-45-1b and Glycocaulis sp. 6B-8 using an electron microscope.

(3) FIG. 3 shows the non-contact culture results of a Glycocaulis sp. 6B-8 suspension and a Dietzia sp. DQ12-45-1b suspension.

(4) FIG. 4A shows the effect of various Glycocaulis strains on the surface motility of alkane-degrading Dietzia sp. DQ12-45-1b; and FIG. 4B shows the effect of Glycocaulis sp. 6B-8 on various alkane-degrading bacteria.

DETAILED DESCRIPTION OF EMBODIMENTS

(5) Unless otherwise specified, the experimental methods employed in the following embodiments are conventional methods.

(6) Unless otherwise specified, the materials and reagents used in the following embodiments are all commercially available.

Example 1

(7) Alkane-degrading Dietzia sp. DQ12-45-1b and Glycocaulis sp. 6B-8 isolated from Daqing oilfield were selected herein as research objects, where Dietzia sp. DQ12-45-1b had been specifically described in “Detection of Metabolites from n-Hexadecane Degradation by Strain Dietzia sp. DQ12-45-1b”, and Glycocaulis sp. 6B-8 had been recorded in “Glycocaulis alkaliphilus sp. nov., a dimorphic prosthecate bacterium isolated from crude oil” (abbreviated as 6B-8).

(8) 1. Investigation on Motility of Dietzia sp. DQ12-45-1b and Glycocaulis sp. 6B-8

(9) 1.1 Preparation of Dietzia sp. DQ12-45-1b and Glycocaulis sp. 6B-8 Suspensions

(10) (1) Preparation of Dietzia sp. DQ12-45-1b Suspension

(11) The strain Dietzia sp. DQ12-45-1b was transferred from a glycerol tube to a plate for activation, and the monoclonal cells were picked and transferred to a test tube containing liquid medium for culture. Then the bacterial suspension in the test tube was transferred to a 500 mL Erlenmeyer flask containing 300 mL of glucose-peptone-yeast extract (GPY) medium and cultured to obtain a seed solution, where the GPY medium (1 L) consisted of 10 g of peptone, 5 g of yeast extract, 10 g of glucose and water, and was adjusted to pH 8.0.

(12) After cultured to the logarithmic growth phase, the seed solution was centrifuged at 5,000 rpm and 4° C. for 10 min, and the cells were collected, washed twice with a MF medium and suspended with the MF medium to a density of 1×10.sup.7 cells/mL to produce the Dietzia sp. DQ12-45-1b suspension, where the MF medium (1 L) consisted of 17.9 g of Na.sub.2HPO.sub.4.12H.sub.2O, 7.8 g of NaH.sub.2PO.sub.4, 5 g of (NH.sub.4).sub.2SO.sub.4, 5 g of KCl, 10 mL of a microelement solution SL-4 (A304-01, Shanghai Linyuan Biotechnology Co., Ltd), 1 mL of sterile 5% CaCl.sub.2) solution, 1 mL of sterile MgSO.sub.4 solution and water, and was adjusted to pH 8.0.

(13) Glucose-peptone-yeast extract (GPY) medium (1 L) was prepared from 10 g of peptone, 5 g of yeast extract, 10 g of glucose and water, and was adjusted to pH 8.0.

(14) (2) Preparation of Glycocaulis sp. 6B-8 Suspension

(15) The Glycocaulis sp. 6B-8 suspension with a density of 1×10.sup.7 cells/mL was prepared according to the above preparation of the Dietzia sp. DQ12-45-1b suspension.

(16) 1.2 Investigation of Bacterial Motility

(17) Due to the failure in utilizing alkane and glucose as carbon sources to grow, Glycocaulis sp. 6B-8 was culture in a LB medium.

(18) Three groups of experiments were designed.

(19) Group 1 Measurement of Surface Motility of Single Strain

(20) A drop of the Dietzia sp. DQ12-45-1b suspension was spotted on a MF plate containing 0.5% agar with 0.5% hexadecane as the sole carbon source, and the surface motility was measured 24 h later, where a cover of the plate was added with a filter paper adsorbing 0.5% of hexadecane (the size of the filter paper was the same as that of the plate with a diameter of 90 mm) for inverted culture.

(21) A drop of the Glycocaulis sp. 6B-8 suspension was spotted on a LB plate containing 0.5% of agar, and the surface motility was measured 24 h later.

(22) Group 2 Surface Motility of Dietzia sp. DQ12-45-1b Under Co-Culture with Viable Glycocaulis sp. 6B-8 Cells

(23) The Glycocaulis sp. 6B-8 suspension and a solid MF medium (containing 5 g agar per liter of a liquid MF medium) were blended evenly at 55° C. and cooled to form a plate. Then a drop of the Dietzia sp. DQ12-45-1b suspension was spotted on the plate, and a cover of the plate was added with a filter paper adsorbing 1 mL of hexadecane (the size of the filter paper was the same as that of the plate with a diameter of 90 mm) for culture.

(24) Group 3 Surface Motility of Dietzia sp. DQ12-45-1b Under Co-Culture with Dead Glycocaulis sp. 6B-8 Cells

(25) The Glycocaulis sp. 6B-8 suspension was inactivated at high temperature.

(26) The inactivated Glycocaulis sp. 6B-8 suspension and a solid MF medium containing 0.5% agar were blended at 55° C. and cooled to form a plate. A drop of the Dietzia sp. DQ12-45-1b suspension was spotted on the plate, and the plate was added with a filter paper adsorbing hexadecane at its cover and then cultured invertedly.

(27) The plates in the above three groups were cultured for 24 h and then observed for the growth status (colony size) and motility (colonial diffusion size) of each strain.

(28) The motion rate was calculated through dividing the diffusion radius by the growth time.

(29) The results were shown in FIGS. 1A-D, where FIG. 1A: Dietzia sp. DQ12-45-1b; FIG. 1B: Glycocaulis sp. 6B-8; FIG. 1C: Dietzia sp. DQ12-45-1b with viable Glycocaulis sp. 6B-8 cells; and FIG. 1D: Dietzia sp. DQ12-45-1b with dead Glycocaulis sp. 6B-8 cells. In each of FIGS. 1A-D, the upper picture illustrated the colonial growth status, and the lower graph showed the change of colony radius over time.

(30) These figures clearly demonstrated that neither Dietzia sp. DQ12-45-1b nor Glycocaulis sp. 6B-8 exhibited surface motility when cultured individually (FIGS. 1A-B); when co-cultured with viable Glycocaulis sp. 6B-8 cells, Dietzia sp. DQ12-45-1b showed surface motility, and the colony diffused at a rate of 2.87±0.28 μm/min (FIG. 1C); while co-cultured with dead Glycocaulis sp. 6B-8 cells, Dietzia sp. DQ12-45-1b failed to show surface motility (FIG. 1D).

(31) It can be concluded based on the above results that the presence of viable Glycocaulis sp. 6B-8 cells induced the occurrence of surface motility in Dietzia sp. DQ12-45-1b.

(32) 1.3 Investigation on Interaction Between Dietzia sp. DQ12-45-1b and Glycocaulis sp. 6B-8

(33) Two experimental models, respectively non-contact culture and contact culture, were designed herein.

(34) Non-Contact Culture

(35) A drop of the Glycocaulis sp. 6B-8 suspension and a drop of the Dietzia sp. DQ12-45-1b were spotted on a solid MF medium (containing 0.5% agar with hexadecane as the sole carbon source) respectively at a distance of 0 cm (indicating that the two drops of suspensions were close to each other, and were separated merely by a microporous membrane), 1 cm and 2 cm. A solid MF medium only spotted with the Dietzia sp. DQ12-45-1b was used as control. A microporous membrane was provided between the two spots to prevent the two kinds of microorganisms from contacting with each other, but the metabolites were allowed to pass through the membrane (the bacterial cells were larger than the pore size (0.22 μm) of the microporous membrane, so they fail to pass through the membrane, but the metabolites were so small in size that they can pass through the membrane).

(36) Contact Culture

(37) A drop of the Glycocaulis sp. 6B-8 suspension and a drop of the Dietzia sp. DQ12-45-1b were spotted on a solid MF medium (containing 0.5% agar with hexadecane as the sole carbon source), where the two spots were close to each other.

(38) After cultured for 24 h, the two strains in the non-contact culture and contact groups were measured for the growth status (colony size) and motility (colonial diffusion size).

(39) It can be seen from FIG. 3 that the two strains of bacteria both can grow in the MF medium, but neither of them exhibited surface motility, which indicated that the non-contact culture failed to cause Dietzia sp. DQ12-45-1b to show motility.

(40) The results of the contact culture group were similar to those shown in FIG. 1C, specifically, the two strains of bacteria both can grow in the MF medium, and Dietzia sp. DQ12-45-1b exhibited motility under the contact with viable Glycocaulis sp. 6B-8 cells, which indicated that the contact culture with the viable Glycocaulis sp. 6B-8 cells enabled the Dietzia sp. DQ12-45-1b to show motility.

(41) The microorganisms in the contact culture group were observed in real time under an electron microscope, and the results were shown in FIG. 2. It was surprisingly found that under contact with the viable Glycocaulis sp. 6B-8 cells, the capsule structure of the alkane-degrading strain Dietzia sp. DQ12-45-1b was broken by the Glycocaulis sp. 6B-8, resulting in the occurrence of a typical darting-motion surface motility in the alkane-degrading strain.

(42) The darting-motion surface motility is a special surface motility, which was derived from the change in bacterial structure caused by the contact of two kinds of bacteria.

(43) 2. Investigation on the Darting-Motion Surface Motility of Alkane-Degrading Bacteria Caused by Glycocaulis Strains

(44) The investigation on the darting-motion surface motility of alkane-degrading bacteria caused by Glycocaulis strains was performed as follows.

(45) A Glycocaulis bacterial suspension and a solid MF medium (containing 5 g of agar per liter of a liquid MF medium) were blended evenly at 55° C. and cooled to form a plate, onto which a drop of the alkane-degrading bacterial suspension was dripped. A cover of the plate was added with a filter paper adsorbing 1 mL of hexadecane, and the plate was cultured invertedly.

(46) In order to further demonstrate the darting-motion surface motility of the alkane-degrading bacteria caused by the Glycocaulis strains, the following experiments were conducted.

(47) 1. Suspensions of Glycocaulis abyssi MCS33.sup.T, Glycocaulis albus SLG210-30A1.sup.T, Glycocaulis sp. 6B-8 and Escherichia coli DH5a were individually blended evenly with a solid MF medium (containing 5 g of agar per liter of a liquid MF medium) at 55° C. and cooled to form a solid plate. Each solid plate was dripped with a drop of the Dietzia sp. DQ12-45-1b suspension, and then cultured invertedly with the cover added with a filter paper adsorbing hexadecane.

(48) A solid MF medium (0.5% agar) spotted with a drop of the Dietzia sp. DQ12-45-1b suspension was used as control.

(49) 2. The Glycocaulis sp. 6B-8 suspension and a solid MF medium (containing 5 g of agar per liter of a liquid MF medium) were blended uniformly at 55° C. and cooled to form a solid plate, which was then spotted with a drop of a Dietzia sp. DQ12-45-1b suspension, a Dietzia psychralcaliphila ILA-1.sup.T suspension and a Dietzia timorensis DSM 45568.sup.T suspension, respectively, and cultured invertedly with the cover added with a filter paper adsorbing hexadecane. The plates free of Glycocaulis sp. 6B-8 were individually spotted with the Dietzia suspensions and used as control.

(50) The above-mentioned Glycocaulis strains (i.e., Glycocaulis abyssi MCS33.sup.T, Glycocaulis albus SLG210-30A1.sup.T and Glycocaulis sp. 6B-8) had been described in Xiang-Lin L, Bai-Sheng X, Man C, et al. (Glycocaulis albus sp. nov. A moderately halophilic dimorphic prosthecate bacterium isolated from petroleum-contaminated saline soil [J]. International Journal of Systematic and Evolutionary Microbiology, 2014, 64(Pt 9)).

(51) The alkane-degrading strain Dietzia psychralcaliphila ILA-1.sup.T had been mentioned in Yumoto I, Nakamura A, Iwata H, et al. (Dietzia psychralcaliphila sp. nov. a novel, facultatively psychrophilic alkaliphile that grows on hydrocarbons [J]. International Journal of Systematic &Evolutionary Microbiology, 2002, 52(Pt1):85). The alkane-degrading strain Dietzia timorensis DSM 45568.sup.T had been mentioned in Bell M E, Bernard K A, Harrington S M, et al. (Lawsonella clevelandensis gen. nov., sp. nov., a new member of the suborder Corynebacterineae isolated from human abscesses[J]. International Journal of Systematic and Evolutionary Microbiology, 2016, 66(8)).

(52) The growth status and motility of the Dietzia strains were detected, and the results were shown in FIGS. 4A-B.

(53) It can be seen from FIG. 4A that the Glycocaulis strains Glycocaulis sp. 6B-8, Glycocaulis abyssi MCS33.sup.T and Glycocaulis albus SLG210-30A1.sup.T all enabled the alkane-degrading strain Dietzia sp. DQ12-45-1b to exhibit surface motility, and the alkane-degrading strain Dietzia sp. DQ12-45-1b did not show surface motility in the presence of the non-Glycocaulis strain Escherichia coli DH5α.

(54) As shown in FIG. 4B, the alkane-degrading strains Dietzia psychralcaliphila ILA-1.sup.T, Dietzia timorensis DSM 45568.sup.T and Dietzia sp. DQ12-45-1b all showed surface motility in the presence of the Glycocaulis strain Glycocaulis sp. 6B-8.

(55) It can be concluded from the above that the Glycocaulis strains enabled the alkane-degrading bacterium to display the surface motility through the interaction therebetween. Therefore, a Glycocaulis strain and an alkane-degrading strain can be employed together in the microbial enhanced oil recovery to expand the microbial migration range and expose more residual oil to the microorganisms, enhancing the microbial oil recovery.

Example 2 Interaction Between a Glycocaulis Strain and an Alkane-Degrading Bacterium in Simulation Experiment

(56) A physical model experiment for enhancing the recovery rate for a natural core was conducted. The natural core had a porosity of 22.6%, a pore volume of 23.8 cm.sup.3 and a water phase permeability of 178×10.sup.−3 μm.sup.2.

(57) The water for injection was added with 1 g/L NH.sub.4H.sub.2PO.sub.4, 1 g/L (NH.sub.4).sub.2SO.sub.4, 1 g/L K.sub.2HPO.sub.4, 1.2 g/L KNO.sub.3, 0.5 g/L peptone and 0.5 g/L yeast extract, and the water for injection was 0.6 times the volume of the natural core.

(58) Three experimental groups (Groups A, B and C) and one control group (Group D) were designed, where group A was added with 1 mL of the Dietzia sp. DQ12-45-1b suspension (10.sup.7 cells/mL); group B was added with 1 mL of the Glycocaulis sp. 6B-8 suspension (10.sup.7 cells/mL); group C was added with 0.5 mL of the Dietzia sp. DQ12-45-1b suspension (10.sup.7 cells/mL) and 0.5 mL of the Glycocaulis sp. 6B-8 suspension (10.sup.7 cells/mL); and group D was without any addition of bacterial suspension.

(59) After cultured statically for 18 h, the core was sliced at an interval of 0.5 cm and washed with sterile water, and the water was collected and spread on a plate. The plate was cultured for 36 h and then observed for the motion of microorganisms.

(60) The results were shown in Table 1, and it can be obtained that the microorganisms in group C, in which 0.5 mL of the Dietzia sp. DQ12-45-1b suspension (10.sup.7 cells/mL) and 0.5 mL of the Glycocaulis sp. 6B-8 suspension (10.sup.7 cells/mL) were added, showed a significantly larger motion distance, indicating that the Glycocaulis strain Glycocaulis sp. 6B-8 enabled the alkane-degrading strain Dietzia sp. DQ12-45-1b to exhibit the motility.

(61) TABLE-US-00001 TABLE 1 Motility test Group Motion distance (cm) A 0.5 B 0.5 C 8.0 D 0