Sulfate-reducing bacteria culture medium and application thereof

Abstract

The application provides a sulfate-reducing bacteria culture medium and application thereof, and components of the culture medium include: a carbon source, a nitrogen source, a phosphorus source, calcium chloride, magnesium chloride, an inorganic sulfur source, an organic sulfur compound with reducibility, inorganic trace elements, agar and water; wherein the mass content of the organic sulfur compound with reducibility is 5-30 mg / L, the mass content of the inorganic trace elements is 152-365 mg / L; the organic sulfur compound with reducibility includes lipoic acid and / or thiamine hydrochloride; and the inorganic trace elements include a combination of zinc elements, manganese elements, molybdenum elements and boron elements. The application introduces inorganic trace elements required by biological cell metabolism and rich sulfur electron acceptor species, promotes the growth and metabolism process of sulfate-reducing bacteria, and enables SRB cells to form visible colonies at a faster speed.

Description

Technical Field

[0001] This invention belongs to the field of gas field water sample detection technology, specifically relating to a sulfate-reducing bacteria culture medium and its application. Background Technology

[0002] Sulfate-reducing bacteria (SRB) are present in oilfield produced water and unconventional gas field flowback fluids. These microorganisms can directly acquire electrons from metals or produce acidic media that corrode metals during their metabolic activities, causing localized metal dissolution and potentially leading to perforation failure in production systems. To effectively prevent microbial corrosion, continuous SRB concentration monitoring is necessary in both oilfields and unconventional gas fields.

[0003] In oil and gas field corrosion control, culture media are commonly used to detect SRB concentration. Currently, many patents related to SRB culture media are for liquid media, such as CN102329851B, CN113881594A, and US20180346955A1, which use the extinction dilution method for detection, judging the presence of SRB based on the phenomenon of SRB metabolism producing sulfides. The inherent drawback of these methods is that the detection cycle is as long as 7 days or more, resulting in a significant delay in the detection results. To improve detection efficiency, it is necessary to explore new rapid detection methods by drawing on the plate counting principle used in the medical and health fields, and to explore the establishment and application of new methods using solid culture media. Currently, commercially available solid culture media generally have a weak effect on promoting SRB cell growth, and the time for forming mature colonies can be as long as 5 days or more. The detection cycle and the cycle of bacterial purification and enrichment cannot be effectively shortened. There is an urgent need for culture media that can promote the rapid growth and reproduction of SRB cells and ensure the required lower limit and accuracy of detection. Summary of the Invention

[0004] To address the aforementioned technical problems, the present invention aims to provide a sulfate-reducing bacteria culture medium and its application.

[0005] To achieve the above objectives, the present invention provides a sulfate-reducing bacteria culture medium, the components of which include:

[0006] Carbon source, nitrogen source, phosphorus source, inorganic salt, inorganic sulfur source, reducing organic sulfide, inorganic trace elements and water; wherein, based on the volume of the culture medium, the mass content of the reducing organic sulfide is 5-30 mg / L and the mass content of the inorganic trace elements is 152-365 mg / L.

[0007] The reducing organic sulfides include lipoic acid and / or thiamine hydrochloride; the inorganic trace elements include a combination of zinc, manganese, molybdenum and boron.

[0008] In this invention, reducing organic sulfides and sulfate are both electron acceptors in the growth and metabolism of sulfate-reducing bacteria (SRB). They are reduced in the reaction to provide energy for SRB, and the reducing organic sulfides play a greater role.

[0009] Preferably, in the above-mentioned sulfate-reducing bacterial culture medium, the reducing organic sulfides include lipoic acid 5-10 mg / L and / or thiamine hydrochloride 10-20 mg / L.

[0010] Preferably, in the above-mentioned sulfate-reducing bacteria culture medium, the inorganic trace elements, based on elemental mass content, include a combination of zinc 50-150 mg / L, manganese 100-200 mg / L, molybdenum 1-5 mg / L, and boron 1-10 mg / L.

[0011] Preferably, in the above-mentioned sulfate-reducing bacteria culture medium, the mass contents of carbon source, nitrogen source, phosphorus source, calcium chloride, magnesium chloride, and agar are as follows: carbon source 10-15 g / L, nitrogen source 1-1.5 g / L, phosphorus source 0.1-0.5 g / L, calcium chloride 0.05-0.08 g / L, magnesium chloride 0.12-0.18 g / L, inorganic sulfur source 1.3-2.1 g / L, and agar 15-20 g / L.

[0012] In the above-mentioned sulfate-reducing bacteria culture medium, preferably, the inorganic sulfur source includes sulfate, more preferably sodium sulfate.

[0013] Preferably, in the above-mentioned sulfate-reducing bacteria culture medium, the carbon source includes sodium lactate.

[0014] Preferably, in the above-mentioned sulfate-reducing bacterial culture medium, the nitrogen source includes tryptone.

[0015] Preferably, in the above-mentioned sulfate-reducing bacteria culture medium, the phosphorus source includes potassium dihydrogen phosphate.

[0016] Preferably, in the above-mentioned sulfate-reducing bacteria culture medium, the substances providing the inorganic trace elements include one or more combinations of ZnCl2, MnCl2, Na2MoO4, and H3BO3.

[0017] Preferably, the sulfate-reducing bacteria culture medium, by mass content, comprises: carbon source, nitrogen source, phosphorus source, calcium chloride, magnesium chloride, sodium sulfate, agar, lipoic acid 5-10 mg / L, thiamine hydrochloride 10-20 mg / L, zinc 50-150 mg / L, manganese 100-200 mg / L, molybdenum 1-5 mg / L, boron 1-10 mg / L, and water as the solvent.

[0018] This invention also provides an application of sulfate-reducing bacteria culture medium in purifying and enriching bacterial strains and detecting the concentration of sulfate-reducing bacteria in unconventional gas field field water.

[0019] The sulfate-reducing bacteria culture medium of the present invention can promote the growth and metabolism of SRB cells, thereby solving the problems of long purification and enrichment cycles and low detection sensitivity of traditional culture media used for SRB detection in oil and gas fields.

[0020] The technical solution provided by this invention has the following beneficial effects:

[0021] This invention promotes the growth and metabolism of SRB cells by introducing inorganic trace elements and a variety of sulfur-containing electron acceptors required for biological cell metabolism. This allows SRB cells to form visible colonies more quickly, shortening the detection cycle and purification enrichment time when analyzing SRB concentration using colony counting methods. Detailed Implementation

[0022] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0023] Example 1

[0024] This embodiment provides a sulfate-reducing bacteria culture medium. Based on the volume of the culture medium and the mass content of each component, the components include: sodium lactate 12 g / L, tryptone 1 g / L, potassium dihydrogen phosphate 0.4 g / L, calcium chloride 0.05 g / L, magnesium chloride 0.15 g / L, sodium sulfate 1.5 g / L, agar 20 g / L, zinc (provided by ZnCl2) 100 mg / L, manganese (provided by MnCl2) 150 mg / L, molybdenum (provided by Na2MoO4) 3 mg / L, boron (provided by H3BO3) 4 mg / L, lipoic acid 6 mg / L, thiamine hydrochloride 15 mg / L, and water as the solvent.

[0025] Example 2

[0026] This embodiment provides a sulfate-reducing bacteria culture medium, which is the same as that in Example 1, except that the mass content of zinc is 50 mg / L, the mass content of manganese is 200 mg / L, the mass content of molybdenum is 5 mg / L, the mass content of boron is 1 mg / L, the mass content of lipoic acid is 10 mg / L, and the mass content of thiamine hydrochloride is 10 mg / L.

[0027] Example 3

[0028] This embodiment provides a sulfate-reducing bacteria culture medium, which is the same as that in Example 1, except that the mass content of zinc is 150 mg / L, the mass content of manganese is 100 mg / L, the mass content of molybdenum is 1 mg / L, the mass content of boron is 10 mg / L, the mass content of lipoic acid is 5 mg / L, and the mass content of thiamine hydrochloride is 20 mg / L.

[0029] Example 4

[0030] This embodiment provides a sulfate-reducing bacteria culture medium, which is the same as that in Example 1, except that it does not contain lipoic acid.

[0031] Example 5

[0032] This embodiment provides a sulfate-reducing bacterial culture medium, which is the same as that in Example 1, except that it does not contain thiamine hydrochloride.

[0033] Comparative Example 1

[0034] This comparative example provides a sulfate-reducing bacteria culture medium, which is the same as that in Example 1, except that it does not contain lipoic acid and thiamine hydrochloride.

[0035] Comparative Example 2

[0036] This comparative example provides a sulfate-reducing bacteria culture medium, which is the same as that in Example 1, except that the mass content of lipoic acid is 20 mg / L and the mass content of thiamine hydrochloride is 30 mg / L.

[0037] Comparative Example 3

[0038] This comparative example provides a sulfate-reducing bacteria culture medium, which is the same as that in Example 1, except that it does not contain lipoic acid or thiamine hydrochloride, and is replaced with sodium sulfate with the same sulfur content.

[0039] Comparative Example 4

[0040] This comparative example provides a sulfate-reducing bacteria culture medium, which is the same as that in Example 1, except that it does not contain zinc.

[0041] Comparative Example 5

[0042] This comparative example provides a sulfate-reducing bacteria culture medium, which is the same as that in Example 1, except that it does not contain manganese.

[0043] Comparative Example 6

[0044] This comparative example provides a sulfate-reducing bacteria culture medium, which is the same as that in Example 1, except that it does not contain molybdenum.

[0045] Comparative Example 7

[0046] This comparative example provides a sulfate-reducing bacteria culture medium, which is the same as that in Example 1, except that it does not contain boron.

[0047] The culture effects of the culture media in the above examples and comparative examples were tested. The test method was as follows: the original concentration of SRB was determined to be 6 × 10⁻⁶ after 7 days using the erasure dilution method. 2 The SRB pure bacterial solution was prepared by pipetting 200 μL of the solution onto the surface of the sulfate-reducing bacteria culture medium in the above examples and comparative examples. The medium was then placed in an electric thermostatic incubator for cultivation. Under the same conditions, the number of SRB colonies formed on the surface of each culture medium after one day of growth was observed. The results are shown in Table 1.

[0048] Table 1 shows the number of SRB colonies and corresponding detection results in the culture media of the examples and comparative examples after 1 day of incubation.

[0049]

[0050]

[0051] According to Table 1, comparing the number of SRB colonies formed within one day with the corresponding test results, it can be seen that the addition of inorganic trace elements and organic sulfides can promote the growth and reproduction of sulfate-reducing bacteria and accelerate colony formation. The lack of any one of the key components will slow down the formation of colonies.

[0052] As can be seen from Examples 1-5, the sulfate-reducing bacteria culture medium of the present invention can significantly promote the growth and metabolic processes of SRB cells.

[0053] Comparing Example 1 and Comparative Example 1, it can be seen that the presence of organic sulfur can significantly accelerate the growth of SRB colonies.

[0054] Comparing Example 1 and Comparative Example 2, it can be seen that excessive organic sulfur content actually inhibits the growth of SRB colonies.

[0055] Comparing Example 1 and Comparative Example 3, it can be seen that the synergistic effect of organic sulfur and inorganic sulfur is better than the effect of inorganic sulfur acting alone.

[0056] Comparing Example 1 and Comparative Examples 4-7, it can be seen that the synergistic effect of the four inorganic trace elements zinc, manganese, molybdenum and boron can accelerate the growth of SRB colonies, and the lack of any one of these elements will lead to a significant slowdown in the growth rate of SRB colonies.

Claims

1. A sulfate-reducing bacteria culture medium, comprising: Carbon source, nitrogen source, phosphorus source, calcium chloride, magnesium chloride, inorganic sulfur source, reducing organic sulfide, inorganic trace elements, agar and water; wherein, based on the volume of the culture medium, the mass content of the reducing organic sulfide is 5-30 mg / L and the mass content of the inorganic trace elements is 152-365 mg / L. The reducing organic sulfides include lipoic acid and / or thiamine hydrochloride; the inorganic trace elements include a combination of zinc, manganese, molybdenum and boron.

2. The sulfate-reducing bacteria culture medium according to claim 1, wherein, The reducing organosulfur compounds include lipoic acid 5-10 mg / L and / or thiamine hydrochloride 10-20 mg / L.

3. The sulfate-reducing bacteria culture medium according to claim 1, wherein, The inorganic trace elements, based on their elemental mass content, include a combination of zinc (50-150 mg / L), manganese (100-200 mg / L), molybdenum (1-5 mg / L), and boron (1-10 mg / L).

4. The sulfate-reducing bacteria culture medium according to claim 1, wherein, The mass contents of carbon source, nitrogen source, phosphorus source, calcium chloride, magnesium chloride, and agar are as follows: carbon source 10-15 g / L, nitrogen source 1-1.5 g / L, phosphorus source 0.1-0.5 g / L, calcium chloride 0.05-0.08 g / L, magnesium chloride 0.12-0.18 g / L, inorganic sulfur source 1.3-2.1 g / L, and agar 15-20 g / L.

5. The sulfate-reducing bacteria culture medium according to claim 1, wherein, The inorganic sulfur source includes sulfate.

6. The sulfate-reducing bacteria culture medium according to claim 1, wherein, The carbon source includes sodium lactate.

7. The sulfate-reducing bacteria culture medium according to claim 1, wherein, The nitrogen source includes tryptone.

8. The sulfate-reducing bacteria culture medium according to claim 1, wherein, The phosphorus source includes potassium dihydrogen phosphate.

9. The sulfate-reducing bacteria culture medium according to claim 1, wherein, The components of the sulfate-reducing bacteria culture medium, by mass content, include: carbon source, nitrogen source, phosphorus source, calcium chloride, magnesium chloride, sodium sulfate, agar, lipoic acid 5-10 mg / L, thiamine hydrochloride 10-20 mg / L, zinc 50-150 mg / L, manganese 100-200 mg / L, molybdenum 1-5 mg / L, boron 1-10 mg / L, and water as the solvent.

10. The application of the sulfate-reducing bacteria culture medium according to any one of claims 1-9 in detecting the concentration of sulfate-reducing bacteria in unconventional gas field field water.

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