Sulfate reducing bacteria culture medium and application thereof in culture and detection of sulfate reducing bacteria and detection method of sulfate reducing bacteria

Abstract

The invention relates to the field of gas field water sample detection, and discloses a sulfate reducing bacteria culture medium, application of the sulfate reducing bacteria culture medium in culture and detection of sulfate reducing bacteria and a detection method of the sulfate reducing bacteria. The culture medium contains an electron mediator and L-cysteine; 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. According to the culture medium provided by the invention, the culture time required by strain purification and concentration detection can be remarkably shortened, and the detection efficiency can be remarkably improved.

Description

Technical Field

[0001] The invention relates to the field of gas field water sample detection, and in particular to application 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 application of the culture medium in culturing and detecting sulfate-reducing bacteria, and a detection method for sulfate-reducing bacteria. Background Art

[0002] In the process of unconventional gas production, corrosion caused by the metabolism of sulfate-reducing bacteria (SRB) is common, which leads to the failure of ground gas gathering pipelines and even downhole oil pipes, seriously restricting the safe production of gas fields. In this regard, it is necessary to continue to carry out research and detection on SRB in accordance with the working conditions of unconventional gas media.

[0003] Culture medium is a commonly used medium for studying bacterial growth metabolism, enrichment and purification, and detecting bacterial concentration. Although the common culture medium 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 often reaches 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 realize the rapid purification and detection of SRB. At present, although various types of culture media have been formed, such as CN102329851A and CN113881594A, two types of SRB culture media have been announced, but the time required for SRB growth and reproduction to form stable colonies has not been effectively shortened. 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. Summary of the invention

[0004] The purpose of the present invention is to overcome the problem in the prior art that when sulfate-reducing bacteria are cultured in a sulfate-reducing bacteria culture medium, the colony of sulfate-reducing bacteria grows slowly, the growth time to form a stable colony is long, and the bacterial detection efficiency is low. The present invention provides an application of a mixture of an electron mediator and L-cysteine ​​in promoting the growth of sulfate-reducing bacteria, a sulfate-reducing bacteria culture medium and its application in the cultivation and detection of sulfate-reducing bacteria. The culture medium can enable the sulfate-reducing bacteria colony to grow rapidly, effectively shorten the culture time required for the purification and concentration detection of sulfate-reducing bacteria, and can significantly improve the detection efficiency.

[0005] 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 within 44 hours, preferably within 35 hours; wherein the electron mediator is selected from riboflavin and / or flavin adenine dinucleotide.

[0006] The 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.

[0007] A third aspect of the present invention provides an application of the culture medium provided by the present invention in the cultivation and detection of sulfate-reducing bacteria.

[0008] A fourth aspect of the present invention provides a method for detecting sulfate-reducing bacteria, the method comprising: culturing sulfate-reducing bacteria in a culture medium provided by the present invention, and when the sulfate-reducing bacteria forms 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.

[0009] Through the above technical solution, the beneficial effects of the present invention are:

[0010] 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 formation time of the colony. 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, thereby significantly improving the experimental and detection efficiency.

[0011] The present invention fully considers various nutrients such as carbon source, nitrogen source, phosphorus source and inorganic salts required in the growth and metabolism of SRB cells. The types of formula components are richer and more complete than other culture media, and it is stipulated that the concentration and ratio of each component are controlled within the range suitable for the growth of SRB, thereby providing a suitable growth environment for SRB to a greater extent and promoting the rapid growth and metabolism of SRB.

[0012] Compared with other types of bacteria 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 need to rely on precision equipment readings. It can fully meet the on-site needs of fast, accurate, low lower limit and simplicity. DETAILED DESCRIPTION

[0013] The endpoints and any values ​​of the ranges disclosed in this article 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 endpoint values ​​of each range, the endpoint values ​​of each range and the 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 as specifically disclosed in this article.

[0014] In a first aspect, the present invention provides an application of a combination of an electron mediator and L-cysteine ​​in promoting the growth of sulfate-reducing bacteria, wherein 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 8mg / L, 29mg / 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, 42m g / 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 / 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, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, 10 mg / L, 11 mg / L, 12 mg / L). / L, 13 mg / L, 14 mg / L, 15 mg / 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 in the culture medium is within 44 hours (for example, it can be 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours and 44 hours and any range between any two values), preferably within 35 hours; wherein,

[0015] The electron mediator is selected from riboflavin and / or flavin adenine dinucleotide.

[0016] In the present invention, the inventors tested multiple strains of sulfate-reducing bacteria and found that the combination of electron mediator and L-cysteine ​​had similar effects on promoting the growth of each sulfate-reducing strain.

[0017] The second aspect of the present invention provides a sulfate-reducing bacteria culture medium, 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, 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 , 23mg / L, 24mg / L, 25mg / L, 26mg / L, 27mg / L, 28mg / L, 29mg / 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 / L, 59mg / L, 60mg / L, 65mg / L, 70mg / L, 75mg / L, 80mg / L, 85mg / L, 90mg / L, 95mg / L and 100mg / L and any range between any two values, the L-cysteine ​​content is 1-20mg / L, For example, it can be 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, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L and 20 mg / L and any range between any two values; wherein the electron mediator is selected from riboflavin and / or flavin adenine dinucleotide.

[0018] 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, and significantly improve the experimental and detection efficiency.

[0019] According to the present invention, preferably, the culture medium further comprises a carbon source, a nitrogen source, a phosphorus source and an inorganic salt.

[0020] 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.

[0021] 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.

[0022] According to the present invention, preferably, the phosphorus source is selected from KH2PO4 and / or K2HPO4, preferably KH2PO4.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] The present invention has no particular restriction on the weight ratio of the carbon source, the nitrogen source and the 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).

[0028] 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.

[0029] 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, 0.03g / L, 0.035g / L, 0.04g / L, 0.045g / L, 0.05g / L, 0.055g / L, 0.06g / L, 0.065g / L and 0.07g / L and any range between any two values), and 1-1.6g / L of sodium chloride and / or potassium chloride (for example, 1g / L, 1.1g / L, 1.2g / L, 1.3g / L, 1.4g / L, 1.5g / L and 1.6g / L and any range between any two values).

[0030] In the present invention, the above-mentioned substances may exist in a form containing or not containing bound water when preparing the culture medium, and (NH4)2FeSO4 may be (NH4)2FeSO4·6H2O, and its content in the culture medium is calculated in a form without bound water.

[0031] 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 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.

[0032] According to a preferred embodiment of the present invention, based on the total volume of the culture medium, the culture medium comprises:

[0033] 7-15 g / L of a carbon source, wherein the carbon source is selected from sodium lactate and / or glucose;

[0034] 0.5-1.7 g / L of a nitrogen source, wherein the nitrogen source is selected from yeast powder and / or tryptone;

[0035] 0.1-0.5 g / L of a phosphorus source, wherein the phosphorus source is selected from KH2PO4 and / or K2HPO4;

[0036] Inorganic salts: 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;

[0037] 10-100 mg / L of electron mediators and 1-20 mg / L of L-cysteine.

[0038] The present invention fully considers various nutrients such as carbon source, nitrogen source, phosphorus source and inorganic salts required in the growth and metabolism of SRB cells. The types of formula components are richer and more complete than other culture media, and it is stipulated that the concentration and ratio of each component are controlled within the range suitable for the growth of SRB, thereby providing a suitable growth environment for SRB to a greater extent and promoting the rapid growth and metabolism of SRB.

[0039] Further, based on the total volume of the culture medium, the culture medium comprises:

[0040] 8-10 g / L of a carbon source, wherein the carbon source is selected from sodium lactate and / or glucose;

[0041] 0.8-1 g / L of a nitrogen source, wherein the nitrogen source is selected from yeast powder and / or tryptone;

[0042] 0.2-0.4 g / L of a phosphorus source, wherein the phosphorus source is selected from KH2PO4 and / or K2HPO4;

[0043] 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;

[0044] 35-45mg / L of electron mediators and 5-10mg / L of L-cysteine.

[0045] According to a more preferred embodiment of the present invention, based on the total volume of the culture medium, the culture medium comprises:

[0046] Carbon source: 8-10 g / L sodium lactate;

[0047] Nitrogen source: 0.8-1g / L tryptone;

[0048] Phosphorus source: 0.2-0.4 g / L KH2PO4;

[0049] 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;

[0050] 35-45mg / L riboflavin and 5-10mg / L L-cysteine.

[0051] The culture medium of the present invention can be a liquid culture medium or a solid culture medium. When the culture medium is a liquid culture medium, sulfate-reducing bacteria are cultured in the liquid culture medium, and the liquid containing the bacteria turns black, indicating that sulfate-reducing bacteria grow; when the culture medium is a solid culture medium, sulfate-reducing bacteria are cultured in the solid culture medium, and the growth of sulfate-reducing bacteria is observed by the number of colonies in the solid culture medium. In order to allow bacterial cells to multiply and form colonies visible to the naked eye, preferably, the culture medium is a solid culture medium, and the culture medium further contains agar.

[0052] 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.

[0053] According to the present invention, preferably, based on the total volume of the culture medium, the culture medium comprises:

[0054] Carbon source: 7-15g / L sodium lactate;

[0055] Nitrogen source: 0.5-1.7 g / L tryptone;

[0056] Phosphorus source: 0.1-0.5 g / L KH2PO4;

[0057] 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;

[0058] 10-100mg / L riboflavin and 1-20mg / L L-cysteine.

[0059] Preferably, based on the total volume of the culture medium, the culture medium further comprises 15-20 g / L agar.

[0060] Furthermore, based on the total volume of the culture medium, the culture medium comprises:

[0061] Carbon source: 8-10 g / L sodium lactate;

[0062] Nitrogen source: 0.8-1g / L tryptone;

[0063] Phosphorus source: 0.2-0.4 g / L KH2PO4;

[0064] 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;

[0065] 35-45mg / L riboflavin and 5-10mg / L L-cysteine.

[0066] Preferably, based on the total volume of the culture medium, the culture medium further comprises 15-20 g / L agar.

[0067] In the present invention, the pH value of the culture medium is 6.8-7.3.

[0068] A third aspect of the present invention provides an application of the culture medium provided by the present invention in the cultivation and detection of sulfate-reducing bacteria.

[0069] Compared with other types of bacteria 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 need to rely on precision equipment readings. It can fully meet the on-site needs of fast, accurate, low lower limit and simplicity.

[0070] A fourth aspect of the present invention provides a method for detecting sulfate-reducing bacteria, the method comprising: culturing sulfate-reducing bacteria in a culture medium provided by the present invention, and when the sulfate-reducing bacteria forms 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.

[0071] The present invention will be described in detail below by way of examples. In the following examples, unless otherwise specified, all methods are conventional methods; and the reagents and materials used, unless otherwise specified, can be obtained from commercial sources.

[0072] The following examples are used to illustrate the preparation of sulfate-reducing bacteria culture medium.

[0073] Example 1

[0074] Prepare the culture medium according to the following formula:

[0075] Based on the total volume of the culture medium, the culture medium contains: 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 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 culture medium is recorded as S1.

[0076] Example 2

[0077] The culture medium was prepared according to the formula of Example 1, except that the types of carbon source and nitrogen source were different, glucose was used instead of an equal weight of sodium lactate, and yeast extract powder was used instead of an equal weight of tryptone to obtain a culture medium, which was recorded as S2.

[0078] Example 3

[0079] The culture medium was prepared according to the formula of Example 1, except that the types of carbon source and nitrogen source were different. Specifically, an equal weight of sodium lactate was replaced by a mixture of glucose and sodium lactate in a weight ratio of 1:1, and an equal weight of tryptone was replaced by a mixture of tryptone and yeast extract powder in a weight ratio of 1:1. The culture medium was obtained and recorded as S3.

[0080] Example 4

[0081] The culture medium was prepared according to the formula of Example 1, except that K2HPO4 was used instead of KH2PO4 of equal weight to obtain a culture medium, which was recorded as S4.

[0082] Example 5

[0083] The culture medium was prepared according to the formula of Example 1, except that potassium chloride was used to replace an equal weight of sodium chloride in the inorganic salts to obtain a culture medium, which was recorded as S5.

[0084] Example 6

[0085] The 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 culture medium was obtained and recorded as S6.

[0086] Example 7

[0087] The culture medium was prepared according to the formula of Example 1, except that the amount of each component in the inorganic salt was different. Specifically, the amount of each component in the inorganic salt was: 0.5 g / L of (NH4)2FeSO4·6H2O, 0.13 g / L of magnesium sulfate, 1 g / L of sodium sulfate, 1 g / L of sodium chloride and 0.03 g / L of anhydrous calcium chloride. The culture medium was obtained and recorded as S7.

[0088] Example 8

[0089] The culture medium was prepared according to the formula of Example 1, except that the amount of riboflavin was 35 mg / L. The culture medium was obtained and recorded as S8.

[0090] Example 9

[0091] The culture medium was prepared according to the formula of Example 1, except that the amount of L-cysteine ​​was 5 mg / L. The culture medium was obtained and recorded as S9.

[0092] Example 10

[0093] The culture medium was prepared according to the formula of Example 1, except that the amount of riboflavin was 20 mg / L. The culture medium was obtained and recorded as S10.

[0094] Embodiment 11

[0095] The culture medium was prepared according to the formula of Example 1, except that the amount of riboflavin was 45 mg / L. The culture medium was obtained and recorded as S11.

[0096] Example 12

[0097] The culture medium was prepared according to the formula of Example 1, except that the amount of L-cysteine ​​was 10 mg / L. The culture medium was obtained and recorded as S12.

[0098] Embodiment 13

[0099] The culture medium was prepared according to the formula of Example 1, except that the amount of L-cysteine ​​was 15 mg / L. The culture medium was obtained and recorded as S13.

[0100] Embodiment 14

[0101] The culture medium was prepared according to the formula of Example 1, except that the amount of riboflavin was 50 mg / L. The culture medium was obtained and recorded as S14.

[0102] Comparative Example 1

[0103] The 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 recorded as D1.

[0104] Comparative Example 2

[0105] The 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 recorded as D2.

[0106] Comparative Example 3

[0107] The 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 recorded as D3.

[0108] Comparative Example 4

[0109] The culture medium was prepared according to the formula of Example 1, except that 2,6-anthraquinone disulfonic acid (AQS) was used to replace an equal weight of riboflavin to obtain a culture medium, which was recorded as D4.

[0110] Comparative Example 5

[0111] The culture medium was prepared according to the formula of Example 1, except that mercaptoethanol was used to replace an equal weight of L-cysteine ​​to obtain a culture medium, which was recorded as D5.

[0112] Comparative Example 6

[0113] The culture medium was prepared according to the formula of Example 1, except that vitamin C was used to replace an equal weight of L-cysteine ​​to obtain a culture medium, which was recorded as D6.

[0114] Comparative Example 7

[0115] The culture medium was prepared according to the formula of Example 1, except that the amount of L-cysteine ​​was 25 mg / L. The culture medium was obtained and recorded as D7.

[0116] Comparative Example 8

[0117] The culture medium was prepared according to the formula of Example 1, except that the amount of riboflavin was 110 mg / L. The culture medium was obtained and recorded as D8.

[0118] Test Example 1

[0119] Prepare three copies of each of S1-S14 medium and D1-D6 medium, and record them as 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 detected are all 10 3 Level. The culture solutions of SRB, FB and TGB were diluted 100 times with deionized water, and 20 μL of the diluted water samples to be tested were taken with a pipette and added to the surface of the prepared S1-S14 culture medium and D1-D8 culture medium respectively; the bacterial solution added to the surface of the culture medium was evenly spread on the entire surface of the culture medium with a coating rod, and the entire operation process was completed on the anaerobic operating table; the culture medium after coating was sealed and placed in an electric constant temperature incubator and cultured at 37°C for 72 hours. The culture medium was taken out and the number of colonies formed on the surface of the S1-S14 culture medium and D1-D8 culture medium for culturing SRB, FB and TGB was observed. Each culture medium experiment was repeated 10 times and the average value was taken. The results are shown in Table 1.

[0120] Table 1

[0121] Culture medium Number of SRB colonies, FB colony number, The number of TGB colonies, S1 84 0 0 S2 66 0 0 S3 70 0 4 S4 72 0 0 S5 75 0 3 S6 74 0 0 S7 71 0 3 S8 80 0 0 S9 82 0 0 S10 69 0 0 S11 81 0 0 S12 80 0 0 S13 72 0 0 S14 71 0 0 D1 43 3 3 D2 52 3 2 D3 49 0 0 D4 58 0 0 D5 55 5 6 D6 56 6 3 D7 61 0 0 D8 59 0 0

[0122] From the results in Table 1, it can be seen that the number of colonies of SRB, FB and TGB formed on the surface of the culture medium of each embodiment is significantly different. The number of colonies grown on the surface of the culture medium with sulfate-reducing bacteria added is significantly greater than that of the culture medium with the other two types of bacterial culture solutions added. The concentrations of the culture solutions 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.

[0123] 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 culture media after 72 hours of culture was less than that in S1 culture medium, indicating that the types and amounts of carbon sources, nitrogen sources and phosphorus sources, the composition and amount of inorganic salts, and the amount of riboflavin and L-cysteine ​​all have a great influence on the number of colonies formed by sulfate-reducing bacteria in the culture medium; and a small amount of TGB still grows in S3, S5 and S7 culture media, indicating that the types of carbon sources and nitrogen sources, as well as the composition and amount of inorganic salts affect not only the number of colonies formed by sulfate-reducing bacteria in the culture medium, but also the selectivity of the culture medium for sulfate-reducing bacteria. In the D1 medium without riboflavin and L-cysteine, the D2 medium without L-cysteine, and the D5 and D6 medium with other components replacing L-cysteine, not only the number of sulfate-reducing bacteria colonies formed after 72 hours was small, but the number of FB and TGB colonies also increased to a certain extent, indicating that not adding L-cysteine ​​to the culture medium, or replacing L-cysteine ​​with other components, will not only affect the number of sulfate-reducing bacteria colonies formed, but also reduce the selectivity for sulfate-reducing bacteria. The number of colonies formed by sulfate-reducing bacteria in D3, D4, D7 and D8 culture medium after 72 hours of culture was less than that in S1-S14 culture medium, indicating that not adding electron mediators to the culture medium, or changing the type of electron mediators, or using L-cysteine ​​in an amount greater than 20 mg / L, or using riboflavin in an amount greater than 100 mg / L will affect the number of sulfate-reducing bacteria colonies formed.

[0124] Test Example 2

[0125] 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) culture medium, Postgate's C (PC) culture medium and Modified Baar's (MB) culture medium commonly used at home and abroad were prepared according to the standard formula as controls. 3 The same bacterial solution was dripped and coated on the surface of each culture medium according to the dilution multiple and operation method of Test Example 1, and cultured in an electric constant temperature incubator. The number of colonies was read every hour. The culture time when the number of colonies no longer increased was the time when the number of colonies on the surface of each culture medium grew to form a stable number of colonies. The results are shown in Table 2.

[0126] Table 2

[0127] Culture medium Time to form a stable colony number, h S1 32 S2 43 S3 41 S4 38 S5 40 S6 37 S7 38 S8 34 S9 33 S10 44 S11 34 S12 34 S13 43 S14 38 D1 85 D2 68 D3 76 D4 72 D5 68 D6 62 D7 61 D8 73 PB 96 PC 64 MB 72

[0128] As can be seen from Table 2, the time used for the S1-S14 culture medium of the present invention is 32-44 h, and the time used for the other cultures is not less than 61 h. 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.

[0129] Test Example 3

[0130] 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) culture medium, Postgate's C (PC) culture medium and Modified Baar's (MB) culture medium commonly used at home and abroad were prepared according to standard recipes as controls.

[0131] Take a certain concentration of 10 2 The backflow 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, and each culture medium plate coated with the backflow fluid was cultured in an electric constant temperature incubator for 72 h. One 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.

[0132] Table 3

[0133] Culture medium Sulfate-reducing bacteria count, count / mL S1 <![CDATA[4.5×10 5 ]]> S2 <![CDATA[6×10 4 ]]> S3 <![CDATA[7.5×10 4 ]]> S4 <![CDATA[6.5×10 4 ]]> S5 <![CDATA[3.5×10 4 ]]> S6 <![CDATA[4×10 4 ]]> S7 <![CDATA[3×10 4 ]]> S8 <![CDATA[1.1×10 5 ]]> S9 <![CDATA[1.8×10 5 ]]> S10 <![CDATA[2.5×10 4 ]]> S11 <![CDATA[1.9×10 5 ]]> S12 <![CDATA[1.8×10 5 ]]> S13 <![CDATA[7×10 4 ]]> S14 <![CDATA[4.5×10 4 ]]> D1 <![CDATA[9.5×10 2 ]]> D2 <![CDATA[8×10 3 ]]> D3 <![CDATA[4.5×10 3 ]]> D4 <![CDATA[3×10 3 ]]> D5 <![CDATA[6.5×10 3 ]]> D6 <![CDATA[8×10 3 ]]> D7 <![CDATA[7×10 3 ]]> D8 <![CDATA[6×10 3 <!-- 10 -->]]> PB <![CDATA[1.8×10 3 ]]> PC <![CDATA[1.1×10 3 ]]> MB <![CDATA[1.5×10 3 ]]>

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

[0135] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope 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 within 44 hours, preferably within 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 content of the nitrogen source in the culture medium 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.