WO2025010976A1 - 硫酸盐还原菌培养基及其在硫酸盐还原菌的培养和检测中的用途和硫酸盐还原菌的检测方法 - Google Patents

硫酸盐还原菌培养基及其在硫酸盐还原菌的培养和检测中的用途和硫酸盐还原菌的检测方法 Download PDF

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WO2025010976A1
WO2025010976A1 PCT/CN2024/071193 CN2024071193W WO2025010976A1 WO 2025010976 A1 WO2025010976 A1 WO 2025010976A1 CN 2024071193 W CN2024071193 W CN 2024071193W WO 2025010976 A1 WO2025010976 A1 WO 2025010976A1
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culture medium
sulfate
reducing bacteria
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cysteine
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French (fr)
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王彦然
刘海峰
余华利
唐永帆
闫静
王智
彭林彩
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Petrochina Co Ltd
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Petrochina Co Ltd
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/045—Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination

Definitions

  • the 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.
  • SRB sulfate-reducing bacteria
  • Culture medium is a commonly used medium for studying bacterial growth metabolism, enrichment and purification, and detection of 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.
  • 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 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 medium and the use of the culture medium in the culture and detection of sulfate-reducing bacteria.
  • the culture medium can enable the sulfate-reducing bacteria colonies 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a fourth aspect of the present invention provides a method for detecting sulfate-reducing bacteria, the method comprising:
  • sulfate-reducing bacteria are cultured in the culture medium provided by the present invention, and when the sulfate-reducing bacteria form a stable colony number in the culture medium, the colony number is read to obtain the concentration of sulfate-reducing bacteria in the sample to be tested.
  • 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.
  • 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.
  • the detection method is based on the growth and cultivation of bacteria in the culture medium provided by the present invention
  • the culture medium provided by the present invention is used for detection, 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.
  • the present invention provides a combination of an electron mediator and L-cysteine for use 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 9mg/L, 30mg/L, 31mg/L, 31mg/L
  • the time for sulfate-reducing bacteria to form a stable colony number in 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,
  • the electron mediator is selected from riboflavin and/or flavin adenine dinucleotide.
  • 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.
  • 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, 11mg/L, 12mg/L, 13mg/L, 14mg/L, 15mg/L, 16mg/L, 17mg/L, 18mg/L, 19mg/L, 20mg/L, 21mg/L, 22mg/L, 23mg/L, 24mg/L, 25mg/L, 26mg/L, 27mg/L, 28mg/L, 29mg/L, 30mg/L, 31mg/L, 32mg/L, 3 3mg/L, 34mg/L, 35mg/L, 36mg/L, 37mg/L, 38mg/L, 39mg/L, 40mg/L,
  • 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.
  • the culture medium further comprises a carbon source, a nitrogen source, a phosphorus source and an inorganic salt.
  • the present invention has no particular limitation on the type of carbon source.
  • the carbon source is selected from sodium lactate and/or glucose, more preferably sodium lactate.
  • the present invention has no particular limitation on the type of nitrogen source.
  • the nitrogen source is selected from yeast powder and/or tryptone, more preferably tryptone.
  • the phosphorus source is selected from KH 2 PO 4 and/or K 2 HPO 4 , preferably KH 2 PO 4 .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the weight ratio of the carbon source, the nitrogen source and the phosphorus source is 10: (0.8-1.2): (0.1-0.4).
  • the inorganic salt comprises sodium sulfate, ferrous ammonium sulfate, magnesium sulfate and calcium chloride, as well as sodium chloride and/or potassium chloride.
  • the culture medium includes: 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, 1.7 g/L, 1.75 g/L, 1.8 g/L, 1.85 g/L, 1.9 g/L, 1.95 g/L and 2 g/L and any range between any two values), 0.5-2 g/L of (NH 4 ) 2 FeSO 4 (for example, it can be 0.5g/L, 0.6
  • the above substances may exist in a form containing or not containing bound water when preparing the culture medium.
  • (NH 4 ) 2 FeSO 4 may be (NH 4 ) 2 FeSO 4 ⁇ 6H 2 O, and its content in the culture medium is calculated in a form containing no bound water.
  • the culture medium comprises: 1.3-1.7 g/L sodium sulfate, 1-1.5 g/L (NH 4 ) 2 FeSO 4 , 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.
  • the culture medium comprises:
  • a carbon source selected from sodium lactate and/or glucose
  • a nitrogen source selected from yeast powder and/or tryptone
  • a phosphorus source selected from KH 2 PO 4 and/or K 2 HPO 4 ;
  • Inorganic salts 0.8-2 g/L sodium sulfate, 0.5-2 g/L (NH 4 ) 2 FeSO 4 , 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;
  • 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.
  • the culture medium comprises:
  • a carbon source 8-10 g/L of a carbon source, wherein the carbon source is selected from sodium lactate and/or glucose;
  • 0.8-1 g/L of a nitrogen source wherein the nitrogen source is selected from yeast powder and/or tryptone;
  • a phosphorus source selected from KH 2 PO 4 and/or K 2 HPO 4 ;
  • Inorganic salts 1.3-1.7 g/L sodium sulfate, 1-1.5 g/L (NH 4 ) 2 FeSO 4 , 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;
  • 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 KH 2 PO 4 ;
  • Inorganic salts 1.3-1.7 g/L sodium sulfate, 1-1.5 g/L (NH 4 ) 2 FeSO 4 , 0.15-0.19 g/L magnesium sulfate, 0.04-0.06 g/L calcium chloride, and 1.2-1.4 g/L chlorine Sodium chloride;
  • the culture medium of the present invention can be a liquid culture medium or a solid culture medium.
  • 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;
  • 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.
  • the culture medium is a solid culture medium, and the culture medium further contains agar.
  • the content of agar in the culture medium is 15-20 g/L.
  • 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 KH 2 PO 4 ;
  • Inorganic salts 0.8-2 g/L sodium sulfate, 0.5-2 g/L (NH 4 ) 2 FeSO 4 , 0.1-0.2 g/L magnesium sulfate, 0.03-0.07 g/L calcium chloride, and 1-1.6 g/L sodium chloride;
  • the culture medium further comprises 15-20 g/L agar.
  • 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 KH 2 PO 4 ;
  • Inorganic salts 1.3-1.7 g/L sodium sulfate, 1-1.5 g/L (NH 4 ) 2 FeSO 4 , 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;
  • the culture medium further comprises 15-20 g/L agar.
  • the pH value of the culture medium is 6.8-7.3.
  • 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.
  • the detection method is based on the growth and cultivation of bacteria in the culture medium provided by the present invention
  • the culture medium provided by the present invention is used for detection, 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.
  • 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.
  • the culture medium contains: 9 g/L sodium lactate, 0.9 g/L tryptone, 1.8 g/L (NH 4 ) 2 FeSO 4 ⁇ 6H 2 O, 1.5 g/L sodium sulfate, 0.3 g/L KH 2 PO 4 , 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.
  • 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.
  • the culture medium was prepared according to the formula of Example 1, except that K 2 HPO 4 was used instead of KH 2 PO 4 of equal weight to obtain a culture medium, which was recorded as S4.
  • 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.
  • 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.
  • the culture medium was prepared according to the formula of Example 1, except that the dosage of each component in the inorganic salt was different. Specifically, the dosage of each component in the inorganic salt was: 0.5 g/L (NH 4 ) 2 FeSO 4 ⁇ 6H 2 O, 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 culture medium was obtained and recorded as S7.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the culture medium was prepared according to the formula of Example 1, except that 2,6-anthraquinone disulfone was selected Acid (AQS) replaced an equal weight of riboflavin to obtain a culture medium, which was recorded as D4.
  • AQS Acid
  • 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.
  • 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.
  • 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.
  • 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.
  • S1-S14 culture medium and D1-D6 culture medium Prepare three copies of S1-S14 culture medium and D1-D6 culture medium respectively, 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
  • FB iron bacteria
  • TGB saprophytic bacteria
  • concentrations of the culture solutions of SRB, FB and TGB to be detected are all at the 10 3 level.
  • 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.
  • 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.
  • 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.
  • the culture media S1-S14 and D1-D8 were prepared according to the formulas of Examples 1-14 and Comparative Examples 1-8, respectively.
  • 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.
  • the bacterial solution with a sulfate-reducing bacteria concentration of 10 3 in Test Example 1 was taken, and the same bacterial solution was dripped and coated on each culture medium according to the dilution multiple and operation method of Test Example 1.
  • the culture medium was placed on the surface of the culture medium 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.
  • 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.
  • Culture media S1-S14 and D1-D8 were prepared according to the formulas 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 formulas as controls.
  • Postgate's B (PB) culture medium Postgate's C (PC) culture medium
  • Modified Baar's (MB) culture medium commonly used at home and abroad were prepared according to standard formulas as controls.
  • a certain amount of 102 concentration of 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, and each culture medium plate coated with the flowback fluid was cultured in an electric constant temperature incubator for 72 h.
  • One of the largest colonies visible to the naked eye in 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.

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Abstract

硫酸盐还原菌培养基及其在硫酸盐还原菌的培养和检测中的用途和硫酸盐还原菌的检测方法。所述培养基包含电子中介物和L-半胱氨酸；以所述培养基的总体积为基准，所述培养基中，所述电子中介物的含量为10-100mg/L，L-半胱氨酸的含量为1-20mg/L；其中，所述电子中介物选自核黄素和/或黄素腺嘌呤二核苷酸。本发明提供的培养基能够显著缩短菌种纯化和浓度检测所需的培养时间，能够显著提升检测效率。

Description

硫酸盐还原菌培养基及其在硫酸盐还原菌的培养和检测中的用途和硫酸盐还原菌的检测方法
相关申请的交叉引用
本申请要求2023年07月13日提交的中国专利申请202310859409.8的权益，该申请的内容通过引用被合并于本文。
技术领域
本发明涉及气田水样检测领域，具体涉及电子中介物和L-半胱氨酸的组合在促进硫酸盐还原菌生长中的用途、一种硫酸盐还原菌培养基及其在硫酸盐还原菌的培养和检测中的用途和硫酸盐还原菌的检测方法。
背景技术
非常规气开采过程中普遍存在硫酸盐还原菌(SRB)代谢造成的腐蚀问题，导致地面集气管线甚至井下油管穿孔失效，严重制约气田安全上产。对此，有必要针对非常规气介质工况，持续开展有关SRB的研究和检测。
培养基是研究细菌生长代谢、富集提纯和检测细菌浓度的常用介质，国内外目前常见的培养基虽然能够有效完成细菌的生长、富集和检测，但是，形成稳定数目的菌落所需的培养时间往往达到3-5天以上，导致实验周期较长，细菌检测的效率低下，关于SRB方面的研究和支撑工作难以高效开展。因此，有必要创新形成能够促进SRB菌落快速生长的培养基，缩短形成稳定菌落的生长时间，实现SRB的快速提纯和检测。当前，虽然已形成了多种类型的培养基，例如CN102329851A和CN113881594A公布了两类SRB培养基，但并未 有效缩短SRB生长繁殖形成稳定菌落所需的时间。因此，国内外的实验研究和细菌检测都亟需能够进一步缩短SRB培养时间，促进SRB快速生长繁殖的培养基，以提高室内科研实验和工业现场细菌检测效率。
发明内容
本发明的目的是为了克服现有技术存在的硫酸盐还原菌培养基培养硫酸盐还原菌时，硫酸盐还原菌菌落生长较慢，形成稳定菌落的生长时间较长，导致细菌检测效率低下的问题，提供电子中介物和L-半胱氨酸的混合物在促进硫酸盐还原菌生长中的用途、一种硫酸盐还原菌培养基及其在硫酸盐还原菌的培养和检测中的用途，该培养基能够使硫酸盐还原菌菌落快速生长，有效缩短硫酸盐还原菌纯化和浓度检测所需的培养时间，能够显著提升检测效率。
为了实现上述目的，本发明第一方面提供电子中介物和L-半胱氨酸的组合在促进硫酸盐还原菌生长中的用途，当培养基中所述电子中介物的含量为10-100mg/L，L-半胱氨酸的含量为1-20mg/L时，硫酸盐还原菌在所述培养基形成稳定菌落数目的时间在24-44h，优选为30-35h；其中，所述电子中介物选自核黄素和/或黄素腺嘌呤二核苷酸。
本发明第二方面提供一种硫酸盐还原菌培养基，所述培养基包含电子中介物和L-半胱氨酸；以所述培养基的总体积为基准，所述培养基中，所述电子中介物的含量为10-100mg/L，L-半胱氨酸的含量为1-20mg/L；其中，所述电子中介物选自核黄素和/或黄素腺嘌呤二核苷酸。
本发明第三方面提供一种本发明提供的培养基在硫酸盐还原菌的培养和检测中的用途。
本发明第四方面提供一种硫酸盐还原菌的检测方法，所述方法包 括：将硫酸盐还原菌在本发明提供的培养基进行培养，当硫酸盐还原菌在所述培养基形成稳定菌落数目时，读取菌落数目，从而得到待测样中硫酸盐还原菌的浓度。
通过上述技术方案，本发明的有益效果为：
本发明通过在培养基组分中加入一定量的电子中介物(核黄素和/或黄素腺嘌呤二核苷酸)及L-半胱氨酸，二者协同作用能够促进SRB的生长和繁殖过程，缩短菌落的形成时间，本发明提供的培养基能够显著缩短菌种纯化和浓度检测所需的培养时间，将利用培养基进行菌种纯化和浓度检测所需的培养时间缩短到44h以内，显著提升了实验和检测效率。
本发明充分考虑SRB细胞生长代谢过程中所需的碳源、氮源、磷源和无机盐等各类营养物质，配方组分类型较其它培养基更加丰富完善，并且规定将各组分的浓度及配比控制在适宜SRB生长的范围内，更大程度地提供SRB适宜的生长环境，促进SRB快速生长代谢。
与其它类型的细菌检测方法比较，因检测方法是基于细菌在本发明提供的培养基中生长培养形成的，所以用途本发明提供的培养基开展检测，检测周期短，能够充分排除死细胞数目的干扰，可广泛用于加注杀菌缓蚀剂前后的水样检测，也不需要依靠精密设备读数，能够充分满足快速、准确、下限低和简便的现场需求。
具体实施方式
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
本发明第一方面提供电子中介物和L-半胱氨酸的组合在促进硫酸盐还原菌生长中的用途，当培养基中所述电子中介物的含量为10-100mg/L(例如可以为10mg/L、11mg/L、12mg/L、13mg/L、14mg/L、15mg/L、16mg/L、17mg/L、18mg/L、19mg/L、20mg/L、21mg/L、22mg/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和100mg/L以及任意两个值之间组成的任意范围)，L-半胱氨酸的含量为1-20mg/L(例如可以为1mg/L、2mg/L、3mg/L、4mg/L、5mg/L、6mg/L、7mg/L、8mg/L、9mg/L、10mg/L、11mg/L、12mg/L、13mg/L、14mg/L、15mg/L、16mg/L、17mg/L、18mg/L、19mg/L和20mg/L以及任意两个值之间组成的任意范围)时，硫酸盐还原菌在所述培养基形成稳定菌落数目的时间在24-44h(例如可以为24h、25h、26h、27h、28h、29h、30h、31h、32h、33h、34h、35h、36h、37h、38h、39h、40h、41h、42h、43h和44h以及任意两个值之间组成的任意范围)，优选为30-35h；其中，
所述电子中介物选自核黄素和/或黄素腺嘌呤二核苷酸。
本发明中，发明人对多株硫酸盐还原菌进行了测试，电子中介物和L-半胱氨酸的组合对促进各硫酸盐还原菌株生长的效果接近。
本发明第二方面提供一种硫酸盐还原菌培养基，所述培养基包含电子中介物和L-半胱氨酸；以所述培养基的总体积为基准，所述培养基中，所述电子中介物的含量为10-100mg/L，例如可以为10mg/L、 11mg/L、12mg/L、13mg/L、14mg/L、15mg/L、16mg/L、17mg/L、18mg/L、19mg/L、20mg/L、21mg/L、22mg/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和100mg/L以及任意两个值之间组成的任意范围，L-半胱氨酸的含量为1-20mg/L，例如可以为1mg/L、2mg/L、3mg/L、4mg/L、5mg/L、6mg/L、7mg/L、8mg/L、9mg/L、10mg/L、11mg/L、12mg/L、13mg/L、14mg/L、15mg/L、16mg/L、17mg/L、18mg/L、19mg/L和20mg/L以及任意两个值之间组成的任意范围；其中，所述电子中介物选自核黄素和/或黄素腺嘌呤二核苷酸。
通过在培养基组分中加入一定量的电子中介物(核黄素和/或黄素腺嘌呤二核苷酸)及L-半胱氨酸，二者协同作用能够促进SRB的生长和繁殖过程，缩短菌落的形成时间，本发明提供的培养基能够显著缩短菌种纯化和浓度检测所需的培养时间，显著提升了实验和检测效率。
根据本发明，优选地，所述培养基还包含碳源、氮源、磷源和无机盐。
本发明对碳源的种类没有特别限制，为了更好地吸收营养，优选地，所述碳源选自乳酸钠和/或葡萄糖，更优选为乳酸钠。
本发明对氮源的种类没有特别限制，为了更好地吸收营养，优选地，所述氮源选自酵母粉和/或胰蛋白胨，更优选为胰蛋白胨。
根据本发明，优选地，所述磷源选自KH2PO4和/或K2HPO4，优选为KH2PO4。
根据本发明，优选地，以所述培养基的总体积为基准，所述培养基中，所述电子中介物的含量为35-45mg/L，L-半胱氨酸的含量为5-10mg/L。
根据本发明，优选地，以所述培养基的总体积为基准，所述培养基中，所述碳源的含量为7-15g/L，例如可以为7g/L、7.5g/L、8g/L、8.5g/L、9g/L、9.5g/L、10g/L、10.5g/L、11g/L、11.5g/L、12g/L、12.5g/L、13g/L、13.5g/L、14g/L、14.5g/L和15g/L以及任意两个值之间组成的任意范围，优选为8-10g/L。
根据本发明，优选地，以所述培养基的总体积为基准，所述培养基中，所述氮源的含量为0.5-1.7g/L，例如可以为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和1.7g/L以及任意两个值之间组成的任意范围，优选为0.8-1g/L。
根据本发明，优选地，以所述培养基的总体积为基准，所述培养基中，所述磷源的含量为0.1-0.5g/L，例如可以为0.1g/L、0.15g/L、0.2g/L、0.25g/L、0.3g/L、0.35g/L、0.4g/L、0.45g/L和0.5g/L以及任意两个值之间组成的任意范围，优选为0.2-0.4g/L。
本发明对培养基中碳源、氮源和磷源的重量比没有特别限制，为了使硫酸盐还原菌细胞生长处于最活跃的状态，优选地，所述培养基中，所述碳源、所述氮源和所述磷源的重量比为10：(0.8-1.2)：(0.1-0.4)。
根据本发明，优选地，所述无机盐包含硫酸钠、硫酸亚铁铵、硫酸镁和氯化钙，以及氯化钠和/或氯化钾。
根据本发明，优选地，以所述培养基的总体积为基准，所述培养 基中包含：0.8-2g/L的硫酸钠(例如可以为0.8g/L、0.85g/L、0.9g/L、0.95g/L、1g/L、1.05g/L、1.1g/L、1.15g/L、1.2g/L、1.25g/L、1.3g/L、1.35g/L、1.4g/L、1.45g/L、1.5g/L、1.55g/L、1.6g/L、1.65g/L、1.7g/L、1.75g/L、1.8g/L、1.85g/L、1.9g/L、1.95g/L和2g/L以及任意两个值之间组成的任意范围)、0.5-2g/L的(NH4)2FeSO4(例如可以为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、1.8g/L、1.9g/L和2g/L以及任意两个值之间组成的任意范围)、0.1-0.2g/L的硫酸镁(例如可以为0.1g/L、0.11g/L、0.12g/L、0.13g/L、0.14g/L、0.15g/L、0.16g/L、0.17g/L、0.18g/L、0.19g/L和0.2g/L以及任意两个值之间组成的任意范围)和0.03-0.07g/L的氯化钙(例如可以为0.03g/L、0.035g/L、0.04g/L、0.045g/L、0.05g/L、0.055g/L、0.06g/L、0.065g/L和0.07g/L以及任意两个值之间组成的任意范围)，以及1-1.6g/L的氯化钠和/或氯化钾(例如可以为1g/L、1.1g/L、1.2g/L、1.3g/L、1.4g/L、1.5g/L和1.6g/L以及任意两个值之间组成的任意范围)。
本发明中，如上所述的物质在配制培养基时，可以以含有或不含有结合水的形式存在，(NH4)2FeSO4可以为(NH4)2FeSO4·6H2O，在所述培养基中以不含结合水的形式计其含量。
根据本发明，优选地，以所述培养基的总体积为基准，所述培养基中包含：1.3-1.7g/L的硫酸钠、1-1.5g/L的(NH4)2FeSO4、0.15-0.19g/L的硫酸镁和0.04-0.06g/L的氯化钙，以及1.2-1.4g/L的氯化钠和/或氯化钾。
根据本发明的一种优选实施方式，以所述培养基的总体积为基准，所述培养基包含：
7-15g/L的碳源，所述碳源选自乳酸钠和/或葡萄糖；
0.5-1.7g/L的氮源，所述氮源选自酵母粉和/或胰蛋白胨；
0.1-0.5g/L的磷源，所述磷源选自KH2PO4和/或K2HPO4；
无机盐：0.8-2g/L的硫酸钠、0.5-2g/L的(NH4)2FeSO4、0.1-0.2g/L的硫酸镁和0.03-0.07g/L的氯化钙，以及1-1.6g/L的氯化钠和/或氯化钾；
10-100mg/L的电子中介物和1-20mg/L的L-半胱氨酸。
本发明充分考虑SRB细胞生长代谢过程中所需的碳源、氮源、磷源和无机盐等各类营养物质，配方组分类型较其它培养基更加丰富完善，并且规定将各组分的浓度及配比控制在适宜SRB生长的范围内，更大程度地提供SRB适宜的生长环境，促进SRB快速生长代谢。
进一步地，以所述培养基的总体积为基准，所述培养基包含：
8-10g/L的碳源，所述碳源选自乳酸钠和/或葡萄糖；
0.8-1g/L的氮源，所述氮源选自酵母粉和/或胰蛋白胨；
0.2-0.4g/L的磷源，所述磷源选自KH2PO4和/或K2HPO4；
无机盐：1.3-1.7g/L的硫酸钠、1-1.5g/L的(NH4)2FeSO4、0.15-0.19g/L的硫酸镁和0.04-0.06g/L的氯化钙，以及1.2-1.4g/L的氯化钠和/或氯化钾；
35-45mg/L的电子中介物和5-10mg/L的L-半胱氨酸。
根据本发明的一种更优选的实施方式，以所述培养基的总体积为基准，所述培养基包含：
碳源：8-10g/L的乳酸钠；
氮源：0.8-1g/L的胰蛋白胨；
磷源：0.2-0.4g/L的KH2PO4；
无机盐：1.3-1.7g/L的硫酸钠、1-1.5g/L的(NH4)2FeSO4、0.15-0.19g/L的硫酸镁和0.04-0.06g/L的氯化钙，以及1.2-1.4g/L的氯 化钠；
35-45mg/L的核黄素和5-10mg/L的L-半胱氨酸。
本发明的培养基可以为液体培养基，也可以为固体培养基。当培养基为液体培养基时，将硫酸盐还原菌在液体培养基中进行培养，含菌培养基液体变为黑色，即表示有硫酸盐还原菌生长；当培养基为固体培养基时，将硫酸盐还原菌在固体培养基中进行培养，通过固体培养基中的菌落数目，观察硫酸盐还原菌的生长情况。为了让细菌细胞繁殖形成肉眼可见的菌落，优选地，所述培养基为固体培养基，所述培养基还包含琼脂。
根据本发明，优选地，以所述培养基的总体积为基准，所述培养基中，琼脂的含量为15-20g/L。
根据本发明，优选地，以所述培养基的总体积为基准，所述培养基包含：
碳源：7-15g/L的乳酸钠；
氮源：0.5-1.7g/L的胰蛋白胨；
磷源：0.1-0.5g/L的KH2PO4；
无机盐：0.8-2g/L的硫酸钠、0.5-2g/L的(NH4)2FeSO4、0.1-0.2g/L的硫酸镁和0.03-0.07g/L的氯化钙，以及1-1.6g/L的氯化钠；
10-100mg/L的核黄素和1-20mg/L的L-半胱氨酸。
优选地，以所述培养基的总体积为基准，所述培养基还包含15-20g/L的琼脂。
进一步地，以所述培养基的总体积为基准，所述培养基包含：
碳源：8-10g/L的乳酸钠；
氮源：0.8-1g/L的胰蛋白胨；
磷源：0.2-0.4g/L的KH2PO4；
无机盐：1.3-1.7g/L的硫酸钠、1-1.5g/L的(NH4)2FeSO4、0.15-0.19g/L的硫酸镁和0.04-0.06g/L的氯化钙，以及1.2-1.4g/L的氯化钠；
35-45mg/L的核黄素和5-10mg/L的L-半胱氨酸。
优选地，以所述培养基的总体积为基准，所述培养基还包含15-20g/L的琼脂。
本发明中，所述培养基的pH值为6.8-7.3。
本发明第三方面提供一种本发明提供的培养基在硫酸盐还原菌的培养和检测中的用途。
与其它类型的细菌检测方法比较，因检测方法是基于细菌在本发明提供的培养基中生长培养形成的，所以用途本发明提供的培养基开展检测，检测周期短，能够充分排除死细胞数目的干扰，可广泛用于加注杀菌缓蚀剂前后的水样检测，也不需要依靠精密设备读数，能够充分满足快速、准确、下限低和简便的现场需求。
本发明第四方面提供一种硫酸盐还原菌的检测方法，所述方法包括：将硫酸盐还原菌在本发明提供的培养基进行培养，当硫酸盐还原菌在所述培养基形成稳定菌落数目时，读取菌落数目，从而得到待测样中硫酸盐还原菌的浓度。
以下将通过实施例对本发明进行详细描述。以下实施例中，如无特殊说明，均为常规方法；所用试剂和材料，如无特殊说明，均可从商业途径获得。
以下实施例用于说明硫酸盐还原菌培养基的配制。
实施例1
按以下配方配制培养基：
以培养基的总体积为基准，培养基包含：9g/L的乳酸钠、0.9g/L的胰蛋白胨、1.8g/L的(NH4)2FeSO4·6H2O、1.5g/L的硫酸钠、0.3g/L的KH2PO4、40mg/L的核黄素、7mg/L的L-半胱氨酸、0.17g/L的硫酸镁、1.3g/L的氯化钠、0.045g/L的无水氯化钙、16g/L的琼脂和去离子水。在121℃的条件下灭菌30min。得到培养基，记为S1。
实施例2
按照实施例1的配方配制培养基，不同的是，碳源和氮源的种类不同，以葡萄糖代替等重量的乳酸钠；以酵母浸粉代替等重量的胰蛋白胨。得到培养基，记为S2。
实施例3
按照实施例1的配方配制培养基，不同的是，碳源和氮源的种类不同。具体地，以重量比为1:1的葡萄糖和乳酸钠的混合物代替等重量的乳酸钠，以重量比为1:1的胰蛋白胨和酵母浸粉的混合物代替等重量的胰蛋白胨。得到培养基，记为S3。
实施例4
按照实施例1的配方配制培养基，不同的是，以K2HPO4代替等重量的KH2PO4。得到培养基，记为S4。
实施例5
按照实施例1的配方配制培养基，不同的是，无机盐中，以氯化钾代替等重量的氯化钠。得到培养基，记为S5。
实施例6
按照实施例1的配方配制培养基，不同的是，乳酸钠和胰蛋白胨的用量不同。具体地，乳酸钠的用量为12g/L，胰蛋白胨的用量为1.5g/L。得到培养基，记为S6。
实施例7
按照实施例1的配方配制培养基，不同的是，无机盐中各组分用量不同。具体地，无机盐中各组分用量为：0.5g/L的(NH4)2FeSO4·6H2O、0.13g/L的硫酸镁、1g/L的硫酸钠、1g/L的氯化钠和0.03g/L的无水氯化钙。得到培养基，记为S7。
实施例8
按照实施例1的配方配制培养基，不同的是，核黄素的用量为35mg/L。得到培养基，记为S8。
实施例9
按照实施例1的配方配制培养基，不同的是，L-半胱氨酸的用量为5mg/L。得到培养基，记为S9。
实施例10
按照实施例1的配方配制培养基，不同的是，核黄素的用量为20mg/L。得到培养基，记为S10。
实施例11
按照实施例1的配方配制培养基，不同的是，核黄素的用量为45mg/L。得到培养基，记为S11。
实施例12
按照实施例1的配方配制培养基，不同的是，L-半胱氨酸的用量为10mg/L。得到培养基，记为S12。
实施例13
按照实施例1的配方配制培养基，不同的是，L-半胱氨酸的用量为15mg/L。得到培养基，记为S13。
实施例14
按照实施例1的配方配制培养基，不同的是，核黄素的用量为50mg/L。得到培养基，记为S14。
对比例1
按照实施例1的配方配制培养基，不同的是，培养基中不添加核黄素和L-半胱氨酸。得到培养基，记为D1。
对比例2
按照实施例1的配方配制培养基，不同的是，培养基中不添加L-半胱氨酸。得到培养基，记为D2。
对比例3
按照实施例1的配方配制培养基，不同的是，培养基中不添加核黄素。得到培养基，记为D3。
对比例4
按照实施例1的配方配制培养基，不同的是，选用2,6-蒽醌二磺 酸(AQS)代替等重量的核黄素。得到培养基，记为D4。
对比例5
按照实施例1的配方配制培养基，不同的是，选用巯基乙醇代替等重量的L-半胱氨酸。得到培养基，记为D5。
对比例6
按照实施例1的配方配制培养基，不同的是，选用维生素C代替等重量的L-半胱氨酸。得到培养基，记为D6。
对比例7
按照实施例1的配方配制培养基，不同的是，L-半胱氨酸的用量为25mg/L。得到培养基，记为D7。
对比例8
按照实施例1的配方配制培养基，不同的是，核黄素的用量为110mg/L。得到培养基，记为D8。
测试例1
将S1-S14培养基和D1-D6培养基各配制三份，依次记为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用于检测还原菌(SRB)在培养基中的生长繁殖情况，#2用于检测铁细菌(FB)在培养基中的生长繁殖情况，#3用于检测腐生菌(TGB)在培养基中的生长繁殖情况。待检测的SRB、FB和TGB的培养液的浓度均为103级。分别将SRB、FB和TGB的培养液用去离子水稀释100倍，再用移液管取20μL稀释后的 待测水样，分别滴加到配制好的S1-S14培养基和D1-D8培养基表面；用涂布棒将滴加在培养基表面的菌液均匀地涂抹在整个培养基表面，整个操作过程在厌氧操作台上完成；将完成涂布的培养基进行密封，放入电热恒温培养箱中，在37℃培养72小时。取出培养基，观察培养SRB、FB和TGB的S1-S14培养基和D1-D8培养基表面形成的菌落数目，每个培养基实验重复进行10次，取平均值，结果如表1所示。
表1
由表1的结果可以看出，各实施例培养基表面形成的SRB、FB和TGB的菌落数目均存在明显的差异，加入硫酸盐还原菌菌液的培养基表面生长出的菌落数目显著大于加入其它两类细菌培养液的培 养基，待检测的SRB、FB和TGB的培养液的浓度均为103级，表明本发明的培养基具有较好的选择性，仅硫酸盐还原菌细胞能够在培养基中正常生长和繁殖，能够特异性地检测硫酸盐还原菌。
其中，硫酸盐还原菌在S2-S7、S10和S13、S14培养基中培养72h后培养基表面形成的菌落数目较S1培养基更少，说明碳源、氮源和磷源的种类和用量，无机盐的组成和用量，核黄素和L-半胱氨酸的用量均对硫酸盐还原菌在培养基形成的菌落数目有较大影响；而S3、S5和S7培养基中还有少量TGB生长，说明碳源和氮源的种类，以及无机盐的组成和用量不仅影响硫酸盐还原菌在培养基形成的菌落数目，还会影响培养基对硫酸盐还原菌的选择性。而不添加核黄素和L-半胱氨酸的D1培养基，不添加L-半胱氨酸的D2培养基，和用其他组分代替L-半胱氨酸的D5和D6，不仅在72h后形成的硫酸盐还原菌菌落数目较少，FB和TGB的菌落数目也有一定程度增加，说明培养基中不添加L-半胱氨酸，或用其他组分代替L-半胱氨酸，不仅会影响硫酸盐还原菌菌落形成的数量，还会降低对硫酸盐还原菌的选择性。硫酸盐还原菌在D3、D4、D7和D8培养基培养72h后形成的菌落数目较在S1-S14培养基中减少，说明培养基中不添加电子中介物，或改变电子中介物的种类，或L-半胱氨酸用量大于20mg/L，或核黄素的用量大于100mg/L会影响硫酸盐还原菌菌落形成的数量。
测试例2
分别按照实施例1-14和对比例1-8的配方配制培养基S1-S14及D1-D8，同时，按照标准配方配制国内外常用的Postgate's B(PB)培养基、Postgate's C(PC)培养基和Modified Baar's(MB)培养基作为对照。取测试例1中硫酸盐还原菌浓度为103级的菌液，按照测试例1的稀释倍数和操作方法，分别将同样的菌液滴加和涂布在各培养基 的表面，并于电热恒温培养箱中培养，每小时读取菌落数目，菌落数目不再增长时所用的培养时间即为各培养基表面生长形成稳定菌落数目的时间。结果如表2所示。
表2
由表2可以看出，本发明的S1-S14培养基所用的时间为32-44h，其余培养所用的时间均不低于61h，由此说明，本发明的培养基能够有效加速硫酸盐还原菌菌落的形成，缩短硫酸盐还原菌纯化和检测时间。
测试例3
分别按照实施例1-14和对比例1-8的配方配制培养基S1-S14及D1-D8，同时，按照标准配方配制国内外常用的Postgate's B(PB)培养基、Postgate's C(PC)培养基和Modified Baar's(MB)培养基作为对照。
取一定量浓度为102级的川南某页岩气田返排液稀释10倍后，分别滴加和涂布在上述培养基平板上，并将涂布有返排液的各培养基平板于电热恒温培养箱中培养72h，挑取各培养基平板中的1个肉眼可见的最大菌落溶于5mL生理盐水中，按照MPN法对生理盐水中的硫酸盐还原菌量进行计数，结果如表3。
表3

由表3可以看出，本发明的S1-S14培养基72h富集得到的单个菌落中的硫酸盐还原菌菌量均大于其余培养基，由此说明，本发明的培养基能够适宜硫酸盐还原菌菌落的快速生长。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于此。在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本发明所公开的内容，均属于本发明的保护范围。

Claims (10)

  1. 电子中介物和L-半胱氨酸的组合在促进硫酸盐还原菌生长中的用途，其特征在于，当培养基中所述电子中介物的含量为10-100mg/L，L-半胱氨酸的含量为1-20mg/L时，硫酸盐还原菌在所述培养基形成稳定菌落数目的时间在24-44h，优选为30-35h；其中，
    所述电子中介物选自核黄素和/或黄素腺嘌呤二核苷酸。
  2. 一种硫酸盐还原菌培养基，其特征在于，所述培养基包含电子中介物和L-半胱氨酸；以所述培养基的总体积为基准，所述培养基中，所述电子中介物的含量为10-100mg/L，L-半胱氨酸的含量为1-20mg/L；其中，所述电子中介物选自核黄素和/或黄素腺嘌呤二核苷酸。
  3. 根据权利要求2所述的培养基，其特征在于，所述培养基还包含碳源、氮源、磷源和无机盐；
    优选地，所述碳源选自乳酸钠和/或葡萄糖，优选为乳酸钠；
    优选地，所述氮源选自酵母粉和/或胰蛋白胨，优选为胰蛋白胨；
    优选地，所述磷源选自KH2PO4和/或K2HPO4，优选为KH2PO4。
  4. 根据权利要求3所述的培养基，其特征在于，以所述培养基的总体积为基准，所述培养基中，所述电子中介物的含量为35-45mg/L，L-半胱氨酸的含量为5-10mg/L；
    优选地，以所述培养基的总体积为基准，所述培养基中，所述碳源的含量为7-15g/L，优选为8-10g/L；
    优选地，以所述培养基的总体积为基准，所述培养基中，所述氮 源的含量为0.5-1.7g/L，优选为0.8-1g/L；
    优选地，以所述培养基的总体积为基准，所述培养基中，所述磷源的含量为0.1-0.5g/L，优选为0.2-0.4g/L。
  5. 根据权利要求3或4所述的培养基，其特征在于，所述无机盐包含硫酸钠、硫酸亚铁铵、硫酸镁和氯化钙，以及氯化钠和/或氯化钾；
    优选地，以所述培养基的总体积为基准，所述培养基中包含：0.8-2g/L的硫酸钠、0.5-2g/L的(NH4)2FeSO4、0.1-0.2g/L的硫酸镁和0.03-0.07g/L的氯化钙，以及1-1.6g/L的氯化钠和/或氯化钾；
    优选地，以所述培养基的总体积为基准，所述培养基中包含：1.3-1.7g/L的硫酸钠、1-1.5g/L的(NH4)2FeSO4、0.15-0.19g/L的硫酸镁和0.04-0.06g/L的氯化钙，以及1.2-1.4g/L的氯化钠和/或氯化钾。
  6. 根据权利要求2-5中任意一项所述的培养基，其特征在于，所述培养基还包含琼脂；
    优选地，以所述培养基的总体积为基准，所述培养基中，琼脂的含量为15-20g/L。
  7. 根据权利要求2-6中任意一项所述的培养基，其特征在于，以所述培养基的总体积为基准，所述培养基包含：
    碳源：7-15g/L的乳酸钠；
    氮源：0.5-1.7g/L的胰蛋白胨；
    磷源：0.1-0.5g/L的KH2PO4；
    无机盐：0.8-2g/L的硫酸钠、0.5-2g/L的(NH4)2FeSO4、0.1-0.2g/L的硫酸镁和0.03-0.07g/L的氯化钙，以及1-1.6g/L的氯化钠；
    10-100mg/L的核黄素和1-20mg/L的L-半胱氨酸；
    优选地，以所述培养基的总体积为基准，所述培养基还包含15-20g/L的琼脂。
  8. 根据权利要求7所述的培养基，其特征在于，以所述培养基的总体积为基准，所述培养基包含：
    碳源：8-10g/L的乳酸钠；
    氮源：0.8-1g/L的胰蛋白胨；
    磷源：0.2-0.4g/L的KH2PO4；
    无机盐：1.3-1.7g/L的硫酸钠、1-1.5g/L的(NH4)2FeSO4、0.15-0.19g/L的硫酸镁和0.04-0.06g/L的氯化钙，以及1.2-1.4g/L的氯化钠；
    35-45mg/L的核黄素和5-10mg/L的L-半胱氨酸；
    优选地，以所述培养基的总体积为基准，所述培养基还包含15-20g/L的琼脂。
  9. 权利要求2-8中任意一项所述的培养基在硫酸盐还原菌的培养和检测中的用途。
  10. 一种硫酸盐还原菌的检测方法，其特征在于，所述方法包括：将硫酸盐还原菌在权利要求2-8中任意一项所述的培养基进行培养，当硫酸盐还原菌在所述培养基形成稳定菌落数目时，读取菌落数目，从而得到待测样中硫酸盐还原菌的浓度。
PCT/CN2024/071193 2023-07-13 2024-01-08 硫酸盐还原菌培养基及其在硫酸盐还原菌的培养和检测中的用途和硫酸盐还原菌的检测方法 Pending WO2025010976A1 (zh)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101570737A (zh) * 2009-06-15 2009-11-04 哈尔滨工业大学 产氢产乙酸细菌互营共培养体的分离筛选方法
WO2014127351A2 (en) * 2013-02-18 2014-08-21 Washington University Compositions and methods to alter gut microbial fermentation using sulfate-reducing bacteria
WO2014140340A1 (en) * 2013-03-14 2014-09-18 Total Research & Technology Feluy Method for production of n-propanol and/or ethanol by fermentation of multiple substrates in a symbiotic manner
CN105899669A (zh) * 2013-03-14 2016-08-24 赛纳塔生物有限公司 通过厌氧微生物的共生共培养物从合成气生产含有n-丙醇和其他C3的产物的方法
CN114634897A (zh) * 2022-04-07 2022-06-17 内蒙古工业大学 降解褐煤的方法及其菌剂

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101570737A (zh) * 2009-06-15 2009-11-04 哈尔滨工业大学 产氢产乙酸细菌互营共培养体的分离筛选方法
WO2014127351A2 (en) * 2013-02-18 2014-08-21 Washington University Compositions and methods to alter gut microbial fermentation using sulfate-reducing bacteria
WO2014140340A1 (en) * 2013-03-14 2014-09-18 Total Research & Technology Feluy Method for production of n-propanol and/or ethanol by fermentation of multiple substrates in a symbiotic manner
CN105899669A (zh) * 2013-03-14 2016-08-24 赛纳塔生物有限公司 通过厌氧微生物的共生共培养物从合成气生产含有n-丙醇和其他C3的产物的方法
CN114634897A (zh) * 2022-04-07 2022-06-17 内蒙古工业大学 降解褐煤的方法及其菌剂

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