Shale gas fracturing flow-back fluid degrading bacterium and application thereof

Abstract

The invention discloses a shale gas fracturing flow-back fluid degrading bacterium and application thereof, and belongs to the technical field of wastewater biological treatment. The shale gas fracturing flow-back fluid degrading bacterium disclosed by the invention is bacillus velezensis Z21-1, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation date is April 23, 2023, and the preservation number is CGMCC NO.27188. The shale gas fracturing flow-back fluid degrading bacterium disclosed by the invention can be used for degrading shale gas fracturing flow-back fluid. The strain can adapt to the growth environment of the shale gas fracturing flow-back fluid, and can efficiently degrade organic matters in the shale gas fracturing flow-back fluid. The invention provides a new strain resource for biological treatment of waste water in the shale gas industry, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of wastewater biological treatment, and specifically relates to shale gas fracturing flowback fluid degradation bacteria and application thereof. Background Art

[0002] Shale gas is an unconventional natural gas that exists in dark mud shale or high-carbon mud shale and exists mainly in adsorption or free state. It has the advantages of cleanliness, high efficiency and long production life. The technically recoverable shale gas resources in my country are 36.1 trillion m 3 The development of shale gas will help reduce my country's dependence on imported energy, promote the optimization of energy structure, increase the proportion of natural gas in energy consumption, and achieve sustainable development goals.

[0003] The hydraulic fracturing technology used in the shale gas mining process can release the methane in the shale layer to the surface, but it will also produce a large amount of flowback fluid. Shale gas flowback fluid contains substances such as alkanes, aromatics, halogenated hydrocarbons, alcohols, esters and ketones, and has the characteristics of high organic matter concentration, complex composition, high salinity and poor biodegradability. For shale gas flowback fluid, the commonly used treatment methods are physical treatment, chemical treatment and biological treatment, among which biological treatment is a green and economical treatment method for shale gas flowback fluid. However, the biodegradability of the flowback fluid is extremely low, the salinity is high, and it contains a large amount of toxic and harmful substances, resulting in low efficiency of conventional biotechnology. Therefore, screening a high-efficiency degradation bacteria suitable for treating shale gas flowback fluid can greatly enhance the application potential of shale gas flowback water biological treatment technology, which is of great significance for serving the green development of the shale gas industry. Summary of the invention

[0004] The purpose of the present invention is to solve the deficiencies in the prior art and provide a shale gas fracturing flowback fluid degradation bacteria and application thereof.

[0005] The specific technical solutions adopted by the present invention are as follows:

[0006] In the first aspect, the present invention provides a shale gas fracturing flowback fluid degrading bacteria, wherein the shale gas fracturing flowback fluid degrading bacteria is Bacillus velezensis Z21-1, which is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with a deposit date of April 23, 2023 and a deposit number of CGMCC NO.27188.

[0007] In a second aspect, the present invention provides an application of the shale gas fracturing flowback fluid degrading bacteria described in the first aspect in the treatment of shale gas fracturing flowback fluid.

[0008] Preferably, the specific method is as follows: the Bacillus velezensis Z21-1 described in claim 1 is cultured in LB liquid culture medium until OD 600 The inoculated bacterial solution is inoculated into the shale gas fracturing flowback fluid at a volume ratio of 1-3%, the reaction temperature is controlled at 30-35°C, the pH is 6-8, and TOC degradation treatment is performed.

[0009] Preferably, the shale gas fracturing flowback fluid includes the flowback fluid formed in the early stage of shale gas production or the flowback fluid formed in the later stage.

[0010] Furthermore, the concentration of suspended particles and organic matter in the initial return liquid is relatively high, and the COD concentration in the initial return liquid is from several thousand to tens of thousands of milligrams per liter. - The concentration ranges from several thousand to tens of thousands of milligrams per liter.

[0011] Furthermore, as the flowback time increases, the organic matter concentration in the flowback liquid formed later decreases and the salinity gradually increases. The COD concentration in the flowback liquid formed later is between several hundred and several thousand milligrams per liter, and the Cl - The concentration is 20000~30000mg / L.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The present invention screens a shale gas fracturing flowback fluid-degrading bacterium from shale gas fracturing flowback fluid, which is identified as Bacillus velezensis. The strain can adapt to the growth environment of shale gas fracturing flowback fluid and can efficiently degrade organic matter in the flowback fluid. It is found through experiments that the TOC degradation rate can reach 52%. The present invention provides a new strain resource for the biological treatment of wastewater in the shale gas industry and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a phylogenetic tree result diagram of Bacillus velezensis Z21-1 screened by the present invention;

[0015] Figure 2 This is a growth curve diagram of Bacillus velezensis Z21-1 screened by the present invention;

[0016] Figure 3 The Bacillus velezensis Z21-1 screened by the present invention was observed under a stereo microscope ( Figure 3 a) Scanning electron microscope ( Figure 3b) Transmission electron microscopy ( Figure 3 c. Figure 3 d) The cell morphology below;

[0017] Figure 4 This is a diagram showing the TOC degradation effect of Bacillus velezensis Z21-1 in Example 2 on rock gas fracturing flowback fluid. DETAILED DESCRIPTION

[0018] The present invention is further described and illustrated below in conjunction with the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly without conflicting with each other.

[0019] Example 1

[0020] This example screens shale gas fracturing flowback fluid degradation bacteria, specifically as follows:

[0021] Take 200 ml of shale gas fracturing flowback fluid in a sterilized conical bottle, seal it and culture it in a shaker at 35°C and 140 rpm for 4-6 hours. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 Perform serial dilutions and take 10 -4 , 10 -5 , 10 -6 100 μL of each of the three gradients was spread on the solid LB medium and cultured in a constant temperature incubator at 30-35°C. The single colony with faster growth on the solid LB medium was picked and placed in 4 mL of liquid LB medium for shaking culture. After 8 hours, the bacterial liquid was dipped and streaked on the solid LB medium for separation. This operation was repeated 4-6 times until a pure single colony was obtained.

[0022] The isolated single colony strain was enriched and cultured and then sent to Shanghai Biotechnology Company for strain identification. The 16SrRNA gene sequence of the strain was used to build a phylogenetic tree using the MEGA program. The results are as follows Figure 1 As shown. Physiological and biochemical identification was performed with reference to the "Common Bacteria System Identification Manual", and the strain was finally identified to the genus. The results showed that the strain was Bacillus, and the similarity with Bacillus velezensis strain YEBBR6 was 99.86%. The obtained strain was named Bacillus velezensis Z21-1.

[0023] The growth curve of Bacillus velezensis Z21-1 in LB liquid medium is as follows Figure 2 As shown in the figure, the stable phase was reached at 8 hours, and the generation time was 1.3 hours. The colony morphology of the above-mentioned Bacillus velezensis Z21-1 was observed using a stereo microscope. Figure 3 a. As can be seen from the figure, the colony is milky white, round, slightly convex, sticky, wrinkled, and has irregular edges.

[0024] The morphology of the above-mentioned Bacillus velezensis Z21-1 was observed by scanning electron microscopy and transmission electron microscopy. Figure 3 b. Figure 3 c. Figure 3 d. As can be seen from the figure, the bacteria are rod-shaped. By performing Gram staining on the strain, it was determined that the bacteria are Gram-positive.

[0025] Example 2

[0026] In this example, Bacillus velezensis Z21-1 obtained by screening in Example 1 was used to degrade TOC in rock gas fracturing flowback fluid, as follows:

[0027] 1) Pick a single colony of Bacillus velezensis Z21-1 on the solid culture medium and place it in LB liquid culture medium, and shake it in a shaker at 35°C and 140 rpm for 8-12 hours. Centrifuge the culture medium at 11000 rpm for 3 minutes, discard the supernatant, and rinse the lower layer with sterilized PBS buffer. Repeat 3 times and adjust the OD 600 To 0.6-0.8 as inoculum solution.

[0028] 2) Take two 100mL portions of shale gas fracturing flowback fluid with a TOC concentration of 450mg / L, the total dissolved solids (TDS) in the water is about 30000mg / L, and Cl - The concentration is about 20000mg / L, BOD 5 / COD is less than 0.1. The components of the shale gas fracturing flowback fluid used include alkanes, aromatic hydrocarbons, halogenated hydrocarbons, alcohols, esters and ketones. Among them, the relative content of branched alkanes and cyclic alkanes is relatively high.

[0029] One portion was inoculated with 1 mL of bacterial solution as the experimental group, and the other portion was not inoculated as the control group. The pH was adjusted to 6-8 and the cells were placed in a shaker at 35°C and 140 rpm for shaking culture.

[0030] 3) The TOC concentration of the experimental group and the control group was measured by TOC analyzer. It was found that the TOC degradation effect of the experimental group was better than that of the control group in the same period of time. The specific results are as follows: Figure 4 shown.

[0031] According to the results, the shale gas fracturing flowback fluid degradation bacteria Bacillus velezensis Z21-1 screened in the example can adapt to the shale gas fracturing flowback fluid environment, and the TOC degradation rate reaches 52% on the 8th day.

[0032] The shale gas fracturing flowback fluid degradation bacteria screened by the present invention can be used to treat the initial flowback fluid and the later flowback fluid. The initial flowback fluid is the flowback fluid formed in the early stage of shale gas extraction, which has high density and viscosity and is black or brown. The concentration of suspended particles and organic matter in the initial flowback fluid is high, and the COD concentration is generally several thousand to tens of thousands of milligrams per liter. - The concentration is several thousand to tens of thousands of milligrams per liter. The residual concentration of chemical additives that have not been completely reacted is relatively high, such as drag reducers, fungicides, clay stabilizers, etc. The late return fluid is the return fluid formed in the early stage of shale gas extraction. As the return time increases, the concentration of organic matter decreases and the salinity gradually increases. The COD concentration in the late return fluid is generally several hundred to several thousand milligrams per liter, and Cl - The concentration is between 20,000 and 30,000 mg / L.

[0033] The above-described embodiment is only a preferred solution of the present invention, but it is not intended to limit the present invention. A person skilled in the relevant technical field may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A shale gas fracturing flowback fluid degradation bacteria, characterized in that: The shale gas fracturing flowback fluid degrading bacteria is Bacillus velezensis Z21-1, which is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration. The deposit date is April 23, 2023, and the deposit number is CGMCCNO.27188.

2. An application of the shale gas fracturing flowback fluid degrading bacteria described in claim 1 in the treatment of shale gas fracturing flowback fluid.

3. The use according to claim 2, characterized in that: The specific method is as follows: the Bacillus velezensis Z21-1 described in claim 1 is cultured in LB liquid medium until OD 600 The inoculated bacterial solution is inoculated into the shale gas fracturing flowback fluid at a volume ratio of 1-3%, the reaction temperature is controlled at 30-35°C, the pH is 6-8, and TOC degradation treatment is performed.

4. The use according to claim 2, characterized in that: The shale gas fracturing flowback fluid includes the flowback fluid formed in the early stage of shale gas production or the flowback fluid formed in the later stage.

5. The use according to claim 4, characterized in that: The concentration of suspended particles and organic matter in the initial return liquid is high. The COD concentration in the initial return liquid is from several thousand to tens of thousands of milligrams per liter. - The concentration ranges from several thousand to tens of thousands of milligrams per liter.

6. The use according to claim 4, characterized in that: As the flowback time increases, the organic matter concentration decreases and the salinity gradually increases. The COD concentration in the flowback liquid formed later is between several hundred and several thousand milligrams per liter, and the Cl - The concentration is 20000~30000mg / L.

Images