Sulfate reducing bacteria culture medium and use thereof in culture and detection of sulfate reducing bacteria, and method for detecting sulfate reducing bacteria

Abstract

A sulfate reducing bacteria culture medium and a use thereof in culture and detection of sulfate reducing bacteria, and a method for detecting sulfate reducing bacteria. The culture medium comprises an electron mediator and L-cysteine; and on the basis of the total volume of the culture medium, in the culture medium, the content of the electron mediator is 10-100 mg / L, and the content of the L-cysteine is 1-20 mg / L, wherein the electron mediator is selected from riboflavin and / or flavin adenine dinucleotide. The culture medium provided by the present invention can significantly shorten the culture time required for strain purification and concentration measurement, and can significantly improve the detection efficiency.

Description

Sulfate-reducing bacteria culture medium and its use in the cultivation and detection of sulfate-reducing bacteria, and sulfate-reducing bacteria detection method

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of Chinese patent application No. 202310859409.8 filed on July 13, 2023, the contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of gas field water sample detection, and in particular to use of a combination of an electron mediator and L-cysteine ​​in promoting the growth of sulfate-reducing bacteria, a sulfate-reducing bacteria culture medium and use of the culture medium in culturing and detecting sulfate-reducing bacteria, and a method for detecting sulfate-reducing bacteria. Background Art

[0004] Corrosion caused by sulfate-reducing bacteria (SRB) metabolism is a common problem in unconventional gas production. This can lead to perforation and failure of surface gas gathering pipelines and even downhole oil pipelines, severely hindering safe gas field production. Therefore, it is necessary to continuously conduct research and testing on SRB in the working conditions of unconventional gas media.

[0005] Culture medium is a commonly used medium for studying bacterial growth metabolism, enrichment and purification, and detecting bacterial concentration. Although the common culture media currently used at home and abroad can effectively complete the growth, enrichment and detection of bacteria, the culture time required to form a stable number of colonies is often more than 3-5 days, resulting in a long experimental cycle and low efficiency of bacterial detection. Research and support work on SRB is difficult to carry out efficiently. Therefore, it is necessary to innovate and form a culture medium that can promote the rapid growth of SRB colonies, shorten the growth time to form stable colonies, and achieve rapid purification and detection of SRB. Currently, although various types of culture media have been formed, such as CN102329851A and CN113881594A, which disclose two types of SRB culture media, they have not effectively shortened the time required for SRB to grow and reproduce to form stable colonies. Therefore, experimental research and bacterial detection at home and abroad are in urgent need of a culture medium that can further shorten the SRB culture time and promote the rapid growth and reproduction of SRB to improve the efficiency of indoor scientific research experiments and industrial field bacterial detection.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to overcome the problem in the prior art that when sulfate-reducing bacteria are cultured in sulfate-reducing bacteria culture media, the sulfate-reducing bacteria colony grows slowly and the growth time to form stable colonies is long, resulting in low bacterial detection efficiency. The present invention provides the use of a mixture of an electron mediator and L-cysteine ​​in promoting the growth of sulfate-reducing bacteria, a sulfate-reducing bacteria culture media and its use in the cultivation and detection of sulfate-reducing bacteria. The culture media can enable sulfate-reducing bacteria colonies to grow rapidly, effectively shorten the culture time required for sulfate-reducing bacteria purification and concentration detection, and significantly improve detection efficiency.

[0008] In order to achieve the above-mentioned purpose, the first aspect of the present invention provides the use of a combination of an electron mediator and L-cysteine ​​in promoting the growth of sulfate-reducing bacteria. When the content of the electron mediator in the culture medium is 10-100 mg / L and the content of L-cysteine ​​is 1-20 mg / L, the time for sulfate-reducing bacteria to form a stable colony number in the culture medium is 24-44 hours, preferably 30-35 hours; wherein, the electron mediator is selected from riboflavin and / or flavin adenine dinucleotide.

[0009] A second aspect of the present invention provides a sulfate-reducing bacteria culture medium, which contains an electron mediator and L-cysteine; based on the total volume of the culture medium, the content of the electron mediator in the culture medium is 10-100 mg / L, and the content of L-cysteine ​​is 1-20 mg / L; wherein the electron mediator is selected from riboflavin and / or flavin adenine dinucleotide.

[0010] A third aspect of the present invention provides a use of the culture medium provided by the present invention in the cultivation and detection of sulfate-reducing bacteria.

[0011] A fourth aspect of the present invention provides a method for detecting sulfate-reducing bacteria, comprising: culturing sulfate-reducing bacteria in a culture medium provided by the present invention; and when the sulfate-reducing bacteria form a stable colony number in the culture medium, reading the colony number to obtain the concentration of sulfate-reducing bacteria in the sample to be tested.

[0012] Through the above technical solution, the beneficial effects of the present invention are:

[0013] The present invention adds a certain amount of electron mediators (riboflavin and / or flavin adenine dinucleotide) and L-cysteine ​​to the culture medium components. The synergistic effect of the two can promote the growth and reproduction process of SRB and shorten the colony formation time. The culture medium provided by the present invention can significantly shorten the culture time required for strain purification and concentration detection, and shortens the culture time required for strain purification and concentration detection using the culture medium to within 44 hours, significantly improving the experimental and detection efficiency.

[0014] The present invention fully considers various nutrients such as carbon sources, nitrogen sources, phosphorus sources and inorganic salts required for the growth and metabolism of SRB cells. The formula component types are richer and more complete than those of other culture media. It also stipulates that the concentration and ratio of each component are controlled within a range suitable for SRB growth, providing a more suitable growth environment for SRB and promoting rapid growth and metabolism of SRB.

[0015] Compared with other types of bacterial detection methods, since the detection method is based on the growth and cultivation of bacteria in the culture medium provided by the present invention, the detection is carried out using the culture medium provided by the present invention, the detection cycle is short, and the interference of the number of dead cells can be fully eliminated. It can be widely used for water sample detection before and after the addition of bactericidal corrosion inhibitors, and does not require reliance on precision equipment for readings, and can fully meet the on-site needs of fast, accurate, low lower limit and simplicity. DETAILED DESCRIPTION

[0016] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0017] In a first aspect of the present invention, an electron mediator and L-cysteine ​​are used in combination to promote the growth of sulfate-reducing bacteria. When the content of the electron mediator in the culture medium is 10-100 mg / L (for example, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L, 20 mg / L, 21 mg / L, 22 mg / L, 23 mg / L, 24 mg / L, 25 mg / L, 26 mg / L, 27 mg / L, 28 mg / L, 29 mg / L, 30 mg / L, 31 mg / L, 32 mg / L, 33 mg / L, 34 mg / L, 35 mg / L, 36 mg / L, 37 mg / L, 38 mg / L, 39 mg / L, 40 mg / L, 41 mg / L, 42 mg / L, 43 mg / L, 44 mg / L, 45 mg / L, 46 mg / L, 47 mg / L, 48 mg / L, 49 mg / L, 50 mg / L, 51 mg / L, 52 mg / L, 53 mg / L, 54 mg / L, 55 mg / L, 56 mg / L, 57 mg / L, 58 mg / L, 59 mg / L, 60 mg / L, 61 mg / L, 62 mg / L, 63 mg / L, 64 mg / L 9mg / L, 30mg / L, 31mg / L, 32mg / L, 33mg / L, 34mg / L, 35mg / L, 36mg / L, 37mg / L, 38mg / L, 39mg / L, 40mg / L, 41mg / L, 42mg / L, 43mg / L , 44mg / L, 45mg / L, 46mg / L, 47mg / L, 48mg / L, 49mg / L, 50mg / L, 51mg / L, 52mg / L, 53mg / L, 54mg / L, 55mg / L, 56mg / L, 57mg / L, 58mg / The content of L-cysteine ​​is 1-20 mg / L (for example, 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, etc.). g / L, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L and 20 mg / L and any range between any two values), the time for sulfate-reducing bacteria to form a stable colony number on the culture medium is 24-44 h (for example, it can be 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, 31 h, 32 h, 33 h, 34 h, 35 h, 36 h, 37 h, 38 h, 39 h, 40 h, 41 h, 42 h, 43 h and 44 h and any range between any two values), preferably 30-35 h; wherein,

[0018] The electron mediator is selected from riboflavin and / or flavin adenine dinucleotide.

[0019] In the present invention, the inventors tested multiple strains of sulfate-reducing bacteria and found that the combination of an electron mediator and L-cysteine ​​had similar effects on promoting the growth of each sulfate-reducing strain.

[0020] A second aspect of the present invention provides a sulfate-reducing bacteria culture medium, the culture medium comprising an electron mediator and L-cysteine; based on the total volume of the culture medium, the content of the electron mediator in the culture medium is 10-100 mg / L, for example, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L, 20 mg / L, 21 mg / L, 22 mg / L, 23 mg / L, 24 mg / L, 25 mg / L, 26 mg / L, 27 mg / L, 28 mg / L, 29 mg / L, 30 mg / L, 31 mg / L, 32 mg / L, 33 mg / L, 34 mg / L, 35 mg / L, 36 mg / L, 37 mg / L, 38 mg / L, 39 mg / L, 40 mg / L, 41 mg / L, 42 mg / L, 43 mg / L, 44 mg / L 3mg / L, 34mg / L, 35mg / L, 36mg / L, 37mg / L, 38mg / L, 39mg / L, 40mg / L, 41mg / L, 42mg / L, 43mg / L, 44 mg / L, 45mg / L, 46mg / L, 47mg / L, 48mg / L, 49mg / L, 50mg / L, 51mg / L, 52mg / L, 53mg / L, 54mg / L, 55m g / L, 56 mg / L, 57 mg / L, 58 mg / L, 59 mg / L, 60 mg / L, 65 mg / L, 70 mg / L, 75 mg / L, 80 mg / L, 85 mg / L, 90 mg / L, 95 mg / L and 100 mg / L and any range between any two values, the content of L-cysteine ​​is 1-20 mg / L, for example, it can be 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 17mg / L, 18mg / L, 19mg / L and 20mg / L, and any range between any two values; wherein the electron mediator is selected from riboflavin and / or flavin adenine dinucleotide.

[0021] By adding a certain amount of electron mediators (riboflavin and / or flavin adenine dinucleotide) and L-cysteine ​​to the culture medium components, the synergistic effect of the two can promote the growth and reproduction process of SRB and shorten the colony formation time. The culture medium provided by the present invention can significantly shorten the culture time required for strain purification and concentration detection, significantly improving experimental and detection efficiency.

[0022] According to the present invention, preferably, the culture medium further comprises a carbon source, a nitrogen source, a phosphorus source and an inorganic salt.

[0023] The present invention has no particular limitation on the type of carbon source. In order to better absorb nutrients, preferably, the carbon source is selected from sodium lactate and / or glucose, more preferably sodium lactate.

[0024] The present invention has no particular limitation on the type of nitrogen source. In order to better absorb nutrients, preferably, the nitrogen source is selected from yeast powder and / or tryptone, more preferably tryptone.

[0025] According to the present invention, preferably, the phosphorus source is selected from KH2PO4 and / or K2HPO4, preferably KH2PO4.

[0026] According to the present invention, preferably, based on the total volume of the culture medium, the content of the electron mediator in the culture medium is 35-45 mg / L, and the content of L-cysteine ​​is 5-10 mg / L.

[0027] According to the present invention, preferably, based on the total volume of the culture medium, the content of the carbon source in the culture medium is 7-15 g / L, for example, it can be 7 g / L, 7.5 g / L, 8 g / L, 8.5 g / L, 9 g / L, 9.5 g / L, 10 g / L, 10.5 g / L, 11 g / L, 11.5 g / L, 12 g / L, 12.5 g / L, 13 g / L, 13.5 g / L, 14 g / L, 14.5 g / L and 15 g / L, and any range between any two values, preferably 8-10 g / L.

[0028] According to the present invention, preferably, based on the total volume of the culture medium, the content of the nitrogen source in the culture medium is 0.5-1.7 g / L, for example, it can be 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L and 1.7 g / L, and any range between any two values, preferably 0.8-1 g / L.

[0029] According to the present invention, preferably, based on the total volume of the culture medium, the content of the phosphorus source in the culture medium is 0.1-0.5 g / L, for example, it can be 0.1 g / L, 0.15 g / L, 0.2 g / L, 0.25 g / L, 0.3 g / L, 0.35 g / L, 0.4 g / L, 0.45 g / L and 0.5 g / L, and any range between any two values, preferably 0.2-0.4 g / L.

[0030] The present invention has no particular restriction on the weight ratio of the carbon source, nitrogen source and phosphorus source in the culture medium. In order to make the sulfate-reducing bacteria cells grow in the most active state, preferably, in the culture medium, the weight ratio of the carbon source, the nitrogen source and the phosphorus source is 10: (0.8-1.2): (0.1-0.4).

[0031] According to the present invention, preferably, the inorganic salt comprises sodium sulfate, ferrous ammonium sulfate, magnesium sulfate and calcium chloride, as well as sodium chloride and / or potassium chloride.

[0032] According to the present invention, preferably, based on the total volume of the culture medium, the culture medium comprises: 0.8-2 g / L of sodium sulfate (for example, 0.8 g / L, 0.85 g / L, 0.9 g / L, 0.95 g / L, 1 g / L, 1.05 g / L, 1.1 g / L, 1.15 g / L, 1.2 g / L, 1.25 g / L, 1.3 g / L, 1.35 g / L, 1.4 g / L, 1.45 g / L, 1.5 g / L, 1.55 g / L, 1.6 g / L, 1.65 g / L). L, 1.7g / L, 1.75g / L, 1.8g / L, 1.85g / L, 1.9g / L, 1.95g / L and 2g / L and any range between any two values), 0.5-2g / L of (NH4)2FeSO4 (for example, 0.5g / L, 0.6g / L, 0.7g / L, 0.8g / L, 0.9g / L, 1g / L, 1.1g / L, 1.2g / L, 1.3g / L, 1.4g / L, 1.5g / L, 1.6g / L, 1.7g / L , 1.8 g / L, 1.9 g / L and 2 g / L and any range between any two values), 0.1-0.2 g / L of magnesium sulfate (for example, 0.1 g / L, 0.11 g / L, 0.12 g / L, 0.13 g / L, 0.14 g / L, 0.15 g / L, 0.16 g / L, 0.17 g / L, 0.18 g / L, 0.19 g / L and 0.2 g / L and any range between any two values), and 0.03-0.07 g / L of calcium chloride (for example, 0.03 g / L, 0.035 g / L, 0.04 g / L, 0.045 g / L, 0.05 g / L, 0.055 g / L, 0.06 g / L, 0.065 g / L and 0.07 g / L and any range consisting of any two values), and 1-1.6 g / L of sodium chloride and / or potassium chloride (for example, 1 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L and 1.6 g / L and any range consisting of any two values).

[0033] In the present invention, the above-mentioned substances may exist in a form containing or not containing bound water when preparing the culture medium. (NH4)2FeSO4 may be (NH4)2FeSO4·6H2O, and its content in the culture medium is calculated as a form containing no bound water.

[0034] According to the present invention, preferably, based on the total volume of the culture medium, the culture medium contains: 1.3-1.7 g / L of sodium sulfate, 1-1.5 g / L of (NH4)2FeSO4, 0.15-0.19 g / L of magnesium sulfate and 0.04-0.06 g / L of calcium chloride, and 1.2-1.4 g / L of sodium chloride and / or potassium chloride.

[0035] According to a preferred embodiment of the present invention, based on the total volume of the culture medium, the culture medium comprises:

[0036] 7-15 g / L of a carbon source, wherein the carbon source is selected from sodium lactate and / or glucose;

[0037] 0.5-1.7 g / L of a nitrogen source, wherein the nitrogen source is selected from yeast powder and / or tryptone;

[0038] 0.1-0.5 g / L of a phosphorus source, wherein the phosphorus source is selected from KH2PO4 and / or K2HPO4;

[0039] Inorganic salts: 0.8-2 g / L sodium sulfate, 0.5-2 g / L (NH4)2FeSO4, 0.1-0.2 g / L magnesium sulfate, 0.03-0.07 g / L calcium chloride, and 1-1.6 g / L sodium chloride and / or potassium chloride;

[0040] 10-100 mg / L of electron mediators and 1-20 mg / L of L-cysteine.

[0041] The present invention fully considers various nutrients such as carbon sources, nitrogen sources, phosphorus sources and inorganic salts required for the growth and metabolism of SRB cells. The formula component types are richer and more complete than those of other culture media. It also stipulates that the concentration and ratio of each component are controlled within a range suitable for SRB growth, providing a more suitable growth environment for SRB and promoting rapid growth and metabolism of SRB.

[0042] Furthermore, based on the total volume of the culture medium, the culture medium comprises:

[0043] 8-10 g / L of a carbon source, wherein the carbon source is selected from sodium lactate and / or glucose;

[0044] 0.8-1 g / L of a nitrogen source, wherein the nitrogen source is selected from yeast powder and / or tryptone;

[0045] 0.2-0.4 g / L of a phosphorus source, wherein the phosphorus source is selected from KH2PO4 and / or K2HPO4;

[0046] Inorganic salts: 1.3-1.7 g / L sodium sulfate, 1-1.5 g / L (NH4)2FeSO4, 0.15-0.19 g / L magnesium sulfate, 0.04-0.06 g / L calcium chloride, and 1.2-1.4 g / L sodium chloride and / or potassium chloride;

[0047] 35-45 mg / L of electron mediators and 5-10 mg / L of L-cysteine.

[0048] According to a more preferred embodiment of the present invention, based on the total volume of the culture medium, the culture medium comprises:

[0049] Carbon source: 8-10 g / L sodium lactate;

[0050] Nitrogen source: 0.8-1 g / L tryptone;

[0051] Phosphorus source: 0.2-0.4 g / L KH2PO4;

[0052] Inorganic salts: 1.3-1.7 g / L sodium sulfate, 1-1.5 g / L (NH4)2FeSO4, 0.15-0.19 g / L magnesium sulfate, 0.04-0.06 g / L calcium chloride, and 1.2-1.4 g / L sodium chloride;

[0053] 35-45mg / L riboflavin and 5-10mg / L L-cysteine.

[0054] The culture medium of the present invention can be either a liquid or solid medium. When the culture medium is a liquid medium, sulfate-reducing bacteria are cultured in the liquid medium. If the liquid medium containing the bacteria turns black, this indicates the growth of sulfate-reducing bacteria. When the culture medium is a solid medium, sulfate-reducing bacteria are cultured in the solid medium, and the growth of the sulfate-reducing bacteria is observed by counting the number of colonies in the solid medium. To allow bacterial cells to reproduce and form visible colonies, the solid medium is preferably a solid medium and further comprises agar.

[0055] According to the present invention, preferably, based on the total volume of the culture medium, the content of agar in the culture medium is 15-20 g / L.

[0056] According to the present invention, preferably, based on the total volume of the culture medium, the culture medium comprises:

[0057] Carbon source: 7-15g / L sodium lactate;

[0058] Nitrogen source: 0.5-1.7 g / L tryptone;

[0059] Phosphorus source: 0.1-0.5 g / L KH2PO4;

[0060] Inorganic salts: 0.8-2g / L sodium sulfate, 0.5-2g / L (NH4)2FeSO4, 0.1-0.2g / L magnesium sulfate, 0.03-0.07g / L calcium chloride, and 1-1.6g / L sodium chloride;

[0061] 10-100mg / L riboflavin and 1-20mg / L L-cysteine.

[0062] Preferably, based on the total volume of the culture medium, the culture medium further comprises 15-20 g / L agar.

[0063] Furthermore, based on the total volume of the culture medium, the culture medium comprises:

[0064] Carbon source: 8-10 g / L sodium lactate;

[0065] Nitrogen source: 0.8-1 g / L tryptone;

[0066] Phosphorus source: 0.2-0.4 g / L KH2PO4;

[0067] Inorganic salts: 1.3-1.7 g / L sodium sulfate, 1-1.5 g / L (NH4)2FeSO4, 0.15-0.19 g / L magnesium sulfate, 0.04-0.06 g / L calcium chloride, and 1.2-1.4 g / L sodium chloride;

[0068] 35-45mg / L riboflavin and 5-10mg / L L-cysteine.

[0069] Preferably, based on the total volume of the culture medium, the culture medium further comprises 15-20 g / L agar.

[0070] In the present invention, the pH value of the culture medium is 6.8-7.3.

[0071] A third aspect of the present invention provides a use of the culture medium provided by the present invention in the cultivation and detection of sulfate-reducing bacteria.

[0072] Compared with other types of bacterial detection methods, since the detection method is based on the growth and cultivation of bacteria in the culture medium provided by the present invention, the detection is carried out using the culture medium provided by the present invention, the detection cycle is short, and the interference of the number of dead cells can be fully eliminated. It can be widely used for water sample detection before and after the addition of bactericidal corrosion inhibitors, and does not require reliance on precision equipment for readings, and can fully meet the on-site needs of fast, accurate, low lower limit and simplicity.

[0073] A fourth aspect of the present invention provides a method for detecting sulfate-reducing bacteria, comprising: culturing sulfate-reducing bacteria in a culture medium provided by the present invention; and when the sulfate-reducing bacteria form a stable colony number in the culture medium, reading the colony number to obtain the concentration of sulfate-reducing bacteria in the sample to be tested.

[0074] The present invention will be described in detail below through examples. In the following examples, unless otherwise specified, conventional methods are used; and the reagents and materials used, unless otherwise specified, can be obtained from commercial sources.

[0075] The following examples are used to illustrate the preparation of sulfate-reducing bacteria culture medium.

[0076] Example 1

[0077] Prepare the culture medium according to the following formula:

[0078] Based on the total volume of the culture medium, the following components were present: 9 g / L sodium lactate, 0.9 g / L tryptone, 1.8 g / L (NH₄)₂FeSO₄·6H₂O, 1.5 g / L sodium sulfate, 0.3 g / L potassium phosphate (KH₂PO₄), 40 mg / L riboflavin, 7 mg / L L-cysteine, 0.17 g / L magnesium sulfate, 1.3 g / L sodium chloride, 0.045 g / L anhydrous calcium chloride, 16 g / L agar, and deionized water. Sterilize at 121°C for 30 min. The resulting culture medium is designated S1.

[0079] Example 2

[0080] A culture medium was prepared according to the recipe of Example 1, except that the carbon and nitrogen sources were different, an equal weight of sodium lactate was replaced by glucose, and an equal weight of tryptone was replaced by yeast extract powder. The resulting culture medium was designated S2.

[0081] Example 3

[0082] A culture medium was prepared according to the recipe of Example 1, except that the carbon and nitrogen sources were different. Specifically, an equal weight of sodium lactate was replaced with a mixture of glucose and sodium lactate at a 1:1 weight ratio, and an equal weight of tryptone was replaced with a mixture of tryptone and yeast extract at a 1:1 weight ratio. The resulting culture medium was designated S3.

[0083] Example 4

[0084] A culture medium was prepared according to the formula of Example 1, except that K2HPO4 was used instead of an equal weight of KH2PO4. The resulting culture medium was designated as S4.

[0085] Example 5

[0086] A culture medium was prepared according to the formula of Example 1, except that potassium chloride was used instead of an equal weight of sodium chloride in the inorganic salts to obtain a culture medium designated as S5.

[0087] Example 6

[0088] A culture medium was prepared according to the formula of Example 1, except that the amounts of sodium lactate and tryptone were different. Specifically, the amount of sodium lactate was 12 g / L and the amount of tryptone was 1.5 g / L. The resulting culture medium was designated S6.

[0089] Example 7

[0090] A culture medium was prepared according to the recipe of Example 1, except that the amounts of the various inorganic salt components were varied. Specifically, the amounts of the inorganic salt components were: 0.5 g / L (NH₄)₂FeSO₄·6H₂O, 0.13 g / L magnesium sulfate, 1 g / L sodium sulfate, 1 g / L sodium chloride, and 0.03 g / L anhydrous calcium chloride. The resulting culture medium was designated S7.

[0091] Example 8

[0092] The culture medium was prepared according to the formula of Example 1, except that the amount of riboflavin was 35 mg / L. The resulting culture medium was designated S8.

[0093] Example 9

[0094] The culture medium was prepared according to the formula of Example 1, except that the amount of L-cysteine ​​was 5 mg / L. The resulting culture medium was designated as S9.

[0095] Example 10

[0096] A culture medium was prepared according to the formula of Example 1, except that the amount of riboflavin was 20 mg / L. The resulting culture medium was designated as S10.

[0097] Example 11

[0098] A culture medium was prepared according to the formula of Example 1, except that the amount of riboflavin was 45 mg / L. The resulting culture medium was designated as S11.

[0099] Example 12

[0100] A culture medium was prepared according to the formula of Example 1, except that the amount of L-cysteine ​​used was 10 mg / L. The resulting culture medium was designated S12.

[0101] Example 13

[0102] A culture medium was prepared according to the formula of Example 1, except that the amount of L-cysteine ​​used was 15 mg / L. The resulting culture medium was designated S13.

[0103] Example 14

[0104] A culture medium was prepared according to the formula of Example 1, except that the amount of riboflavin was 50 mg / L. The resulting culture medium was designated S14.

[0105] Comparative Example 1

[0106] A culture medium was prepared according to the formula of Example 1, except that riboflavin and L-cysteine ​​were not added to the culture medium to obtain a culture medium, which was designated as D1.

[0107] Comparative Example 2

[0108] A culture medium was prepared according to the formula of Example 1, except that L-cysteine ​​was not added to the culture medium to obtain a culture medium, which was designated as D2.

[0109] Comparative Example 3

[0110] A culture medium was prepared according to the formula of Example 1, except that riboflavin was not added to the culture medium to obtain a culture medium, which was designated as D3.

[0111] Comparative Example 4

[0112] A culture medium was prepared according to the formula of Example 1, except that 2,6-anthraquinone disulfonic acid (AQS) was used instead of an equal weight of riboflavin to obtain a culture medium, which was designated as D4.

[0113] Comparative Example 5

[0114] A culture medium was prepared according to the formula of Example 1, except that mercaptoethanol was used instead of an equal weight of L-cysteine ​​to obtain a culture medium designated as D5.

[0115] Comparative Example 6

[0116] A culture medium was prepared according to the formula of Example 1, except that an equal weight of L-cysteine ​​was replaced with vitamin C to obtain a culture medium designated as D6.

[0117] Comparative Example 7

[0118] The culture medium was prepared according to the formula of Example 1, except that the amount of L-cysteine ​​was 25 mg / L. The resulting culture medium was designated as D7.

[0119] Comparative Example 8

[0120] The culture medium was prepared according to the formula of Example 1, except that the amount of riboflavin was 110 mg / L. The resulting culture medium was designated D8.

[0121] Test Example 1

[0122] Prepare three portions of each of S1-S14 culture medium and D1-D6 culture medium, which are named S1#1, S1#2, S1#3, S2#1, S2#2, S2#3, ..., S14#1, S14#2, S14#3, ..., D8#1, D8#2, D8#3. #1 is used to detect the growth and reproduction of reducing bacteria (SRB) in the culture medium, #2 is used to detect the growth and reproduction of iron bacteria (FB) in the culture medium, and #3 is used to detect the growth and reproduction of saprophytic bacteria (TGB) in the culture medium. The concentrations of the culture solutions of SRB, FB, and TGB to be tested are all 10 3 Level. The culture medium of each SRB, FB, and TGB strain was diluted 100-fold with deionized water. A 20 μL sample of the diluted water to be tested was then pipetted onto the surface of the prepared S1-S14 and D1-D8 culture media, respectively. The bacterial solution was evenly spread across the entire surface of the culture medium using a spreading rod. This entire process was performed on an anaerobic workbench. The coated culture medium was sealed and placed in an electrically heated constant-temperature incubator at 37°C for 72 hours. The culture medium was removed and the number of colonies formed on the surface of the S1-S14 and D1-D8 culture media for SRB, FB, and TGB was observed. The experiment was repeated 10 times for each culture medium, and the average results were calculated. Table 1 shows the results.

[0123] Table 1

[0124] From the results in Table 1, it can be seen that there are obvious differences in the number of SRB, FB and TGB colonies formed on the surface of the culture medium of each example. The number of colonies grown on the surface of the culture medium with sulfate-reducing bacteria solution added is significantly greater than that of the culture medium with the other two types of bacteria solution added. The concentrations of the culture medium of SRB, FB and TGB to be tested are all 10 3 The results show that the culture medium of the present invention has good selectivity, only sulfate-reducing bacteria cells can grow and reproduce normally in the culture medium, and sulfate-reducing bacteria can be specifically detected.

[0125] Among them, the number of colonies formed on the surface of the culture medium by sulfate-reducing bacteria in S2-S7, S10, S13 and S14 after culturing for 72 hours was less than that in S1 culture medium, indicating that the type and amount of carbon source, nitrogen source and phosphorus source, the composition and amount of inorganic salts, and the amount of riboflavin and L-cysteine ​​all had a great influence on the number of colonies formed by sulfate-reducing bacteria in the culture medium; while a small amount of TGB still grew in S3, S5 and S7 culture media, indicating that the type of carbon source and nitrogen source, as well as the composition and amount of inorganic salts, not only affected the number of colonies formed by sulfate-reducing bacteria in the culture medium, but also affected the selectivity of the culture medium for sulfate-reducing bacteria. In contrast, D1, D2, and D5 and D6, which lack riboflavin and L-cysteine, not only produced fewer SRB colonies after 72 hours, but also saw a slight increase in the number of FB and TGB colonies. This suggests that omitting L-cysteine ​​or replacing it with other components not only reduces the number of SRB colonies formed but also decreases selectivity for SRB. The number of SRB colonies formed after 72 hours in D3, D4, D7, and D8 media was lower than in S1-S14 media, suggesting that omitting or changing the type of electron mediator, using L-cysteine ​​at a concentration exceeding 20 mg / L, or using riboflavin at a concentration exceeding 100 mg / L can affect the number of SRB colonies formed.

[0126] Test Example 2

[0127] The culture media S1-S14 and D1-D8 were prepared according to the formulas of Examples 1-14 and Comparative Examples 1-8, respectively. At the same time, Postgate's B (PB) medium, Postgate's C (PC) medium and Modified Baar's (MB) medium commonly used at home and abroad were prepared according to the standard formula as controls. 3 The same bacterial solution was dripped and spread onto the surface of each culture medium according to the dilution factor and operation method of Test Example 1. The culture was then incubated in an electric constant-temperature incubator. The colony count was recorded every hour. The incubation time when the colony count stopped increasing was the time it took for the growth of a stable colony count on the surface of each culture medium. The results are shown in Table 2.

[0128] Table 2

[0129] As can be seen from Table 2, the culture medium S1-S14 of the present invention takes 32-44 hours, and the culture time of the other cultures is not less than 61 hours. This shows that the culture medium of the present invention can effectively accelerate the formation of sulfate-reducing bacteria colonies and shorten the purification and detection time of sulfate-reducing bacteria.

[0130] Test Example 3

[0131] Culture media S1-S14 and D1-D8 were prepared according to the recipes of Examples 1-14 and Comparative Examples 1-8, respectively. Meanwhile, Postgate's B (PB) medium, Postgate's C (PC) medium and Modified Baar's (MB) medium commonly used at home and abroad were prepared according to standard recipes as controls.

[0132] Take a certain concentration of 10 2 The flowback fluid from a shale gas field in southern Sichuan was diluted 10 times and then dripped and coated on the above-mentioned culture medium plates, respectively. Each culture medium plate coated with the flowback fluid was incubated in an electric constant temperature incubator for 72 h. The largest colony visible to the naked eye from each culture medium plate was picked and dissolved in 5 mL of normal saline. The amount of sulfate-reducing bacteria in the normal saline was counted according to the MPN method. The results are shown in Table 3.

[0133] Table 3

[0134] As can be seen from Table 3, the amount of sulfate-reducing bacteria in the single colonies obtained by enrichment in the S1-S14 culture media of the present invention for 72 hours is greater than that in the other culture media, which shows that the culture media of the present invention is suitable for the rapid growth of sulfate-reducing bacteria colonies.

[0135] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. Use of a combination of an electron mediator and L-cysteine ​​in promoting the growth of sulfate-reducing bacteria, characterized in that: When the content of the electron mediator in the culture medium is 10-100 mg / L and the content of L-cysteine ​​is 1-20 mg / L, the time for sulfate-reducing bacteria to form a stable colony number in the culture medium is 24-44 hours, preferably 30-35 hours; wherein, The electron mediator is selected from riboflavin and / or flavin adenine dinucleotide.

2. A sulfate-reducing bacteria culture medium, characterized in that: The culture medium contains an electron mediator and L-cysteine; based on the total volume of the culture medium, the content of the electron mediator in the culture medium is 10-100 mg / L, and the content of L-cysteine ​​is 1-20 mg / L; wherein the electron mediator is selected from riboflavin and / or flavin adenine dinucleotide.

3. The culture medium according to claim 2, characterized in that The culture medium also contains a carbon source, a nitrogen source, a phosphorus source and an inorganic salt; Preferably, the carbon source is selected from sodium lactate and / or glucose, preferably sodium lactate; Preferably, the nitrogen source is selected from yeast powder and / or tryptone, preferably tryptone; Preferably, the phosphorus source is selected from KH2PO4 and / or K2HPO4, preferably KH2PO4.

4. The culture medium according to claim 3, characterized in that Based on the total volume of the culture medium, the content of the electron mediator in the culture medium is 35-45 mg / L, and the content of L-cysteine ​​is 5-10 mg / L; Preferably, based on the total volume of the culture medium, the content of the carbon source in the culture medium is 7-15 g / L, preferably 8-10 g / L; Preferably, based on the total volume of the culture medium, the nitrogen in the culture medium The content of the source is 0.5-1.7 g / L, preferably 0.8-1 g / L; Preferably, based on the total volume of the culture medium, the content of the phosphorus source in the culture medium is 0.1-0.5 g / L, preferably 0.2-0.4 g / L.

5. The culture medium according to claim 3 or 4, characterized in that The inorganic salts include sodium sulfate, ammonium ferrous sulfate, magnesium sulfate and calcium chloride, as well as sodium chloride and / or potassium chloride; Preferably, based on the total volume of the culture medium, the culture medium contains: 0.8-2 g / L sodium sulfate, 0.5-2 g / L (NH4)2FeSO4, 0.1-0.2 g / L magnesium sulfate and 0.03-0.07 g / L calcium chloride, and 1-1.6 g / L sodium chloride and / or potassium chloride; Preferably, based on the total volume of the culture medium, the culture medium contains: 1.3-1.7 g / L sodium sulfate, 1-1.5 g / L (NH4)2FeSO4, 0.15-0.19 g / L magnesium sulfate and 0.04-0.06 g / L calcium chloride, and 1.2-1.4 g / L sodium chloride and / or potassium chloride.

6. The culture medium according to any one of claims 2 to 5, characterized in that The culture medium further comprises agar; Preferably, based on the total volume of the culture medium, the content of agar in the culture medium is 15-20 g / L.

7. The culture medium according to any one of claims 2 to 6, characterized in that Based on the total volume of the culture medium, the culture medium comprises: Carbon source: 7-15g / L sodium lactate; Nitrogen source: 0.5-1.7 g / L tryptone; Phosphorus source: 0.1-0.5 g / L KH2PO4; Inorganic salts: 0.8-2g / L sodium sulfate, 0.5-2g / L (NH4)2FeSO4, 0.1-0.2g / L magnesium sulfate, 0.03-0.07g / L calcium chloride, and 1-1.6g / L sodium chloride; 10-100 mg / L riboflavin and 1-20 mg / L L-cysteine; Preferably, based on the total volume of the culture medium, the culture medium further comprises 15-20 g / L agar.

8. The culture medium according to claim 7, characterized in that Based on the total volume of the culture medium, the culture medium comprises: Carbon source: 8-10 g / L sodium lactate; Nitrogen source: 0.8-1g / L tryptone; Phosphorus source: 0.2-0.4 g / L KH2PO4; Inorganic salts: 1.3-1.7 g / L sodium sulfate, 1-1.5 g / L (NH4)2FeSO4, 0.15-0.19 g / L magnesium sulfate, 0.04-0.06 g / L calcium chloride, and 1.2-1.4 g / L sodium chloride; 35-45 mg / L riboflavin and 5-10 mg / L L-cysteine; Preferably, based on the total volume of the culture medium, the culture medium further comprises 15-20 g / L agar.

9. Use of the culture medium according to any one of claims 2 to 8 in the cultivation and detection of sulfate-reducing bacteria.

10. A method for detecting sulfate-reducing bacteria, characterized in that: The method comprises: culturing sulfate-reducing bacteria in the culture medium described in any one of claims 2 to 8, and when the sulfate-reducing bacteria form a stable colony number in the culture medium, reading the colony number to obtain the concentration of sulfate-reducing bacteria in the sample to be tested.

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