CN107937272B - Culture medium for hydrogen sulfide producing bacteria in oil field and application thereof - Google Patents
Culture medium for hydrogen sulfide producing bacteria in oil field and application thereof Download PDFInfo
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Abstract
The invention belongs to the fields of environmental microbiology, microorganism detection and quantity monitoring, and relates to a culture medium formula component suitable for effectively meeting the growth requirement of hydrogen sulfide producing bacteria and promoting the metabolism and the propagation of the hydrogen sulfide producing bacteria and application thereof. The culture medium of the invention consists of basic culture medium solution, trace element solution, vitamin solution and the like, and also contains compound nutrient components such as organic carbon source, sulfur source and the like. Under the anaerobic condition, a proper volume of an anaerobically collected oil field environmental water sample (such as produced water) is inoculated into the culture medium, and the anaerobic culture is carried out under the condition that the in-situ temperature of the environmental sample is the same, so that the growth and metabolism of various microorganism physiological groups with the capability of producing hydrogen sulfide in the sample can be efficiently promoted. By using the culture medium and combining other anaerobic microbiology methods, strain enrichment, separation and screening and microorganism quantity detection and monitoring of hydrogen sulfide producing bacteria in samples of oil field environments or other environmental sources can be effectively realized.
Description
Technical Field
The invention belongs to the fields of environmental microbiology, microorganism detection and quantity monitoring, and relates to a culture medium formula component suitable for effectively meeting the growth requirement of hydrogen sulfide producing bacteria and promoting the metabolism and the propagation of the hydrogen sulfide producing bacteria and application thereof.
Background
In the industrial fields of oil extraction and the like, hydrogen sulfide (H) is generated due to the growth and reproduction of microorganisms2S) brings about a lot of hazards to industrial production: (1) h2S can cause harm to the personal health of personnel and even life safety. When H is present2When the concentration of S is higher (50-200 ppm), the gas poisons human organs due to oxygen deficiency; when the concentration is extremely high (700-2000 ppm), people can lose self-rescue ability only in a few seconds, and the people die if the people are not rescued in time; (2) can generate H2The microorganisms of S can also cause the microbial corrosion of pipelines in the petroleum industry, thereby not only causing great economic loss, but also causing safety and production accidents; (3) High concentration of H2S gas can reduce the economic value of petroleum and natural gas; (4) produce H2The S microorganisms can also cause the oil reservoir stratum to be blocked, so that the oil exploitation difficulty is increased, and the development cost is increased. Therefore, the method can effectively detect the number of the hydrogen sulfide producing bacteria in the oil field environment and monitor the number change of the hydrogen sulfide producing bacteria, and has important value for oil field development industrial enterprises.
For a long time, the culture medium for detecting hydrogen sulfide producing bacteria in oil field enterprises is only directed to sulfate reducing bacteria. However, many recent studies have shown that various kinds of H can be produced in the environment of oil field and the like2S "non-sulfate-reducing bacteria" that are unable to utilize sulfate to produce H2S, but can be generated using a source such as sulfite, thiosulfate, elemental sulfur, or the like2And S. Thus, including sulfate-reducing bacteria, these microbial populations are collectively referred to as hydrogen sulfide producing bacteria. At present, there is a lack of a culture medium suitable for various hydrogen sulfide-producing bacteria. Obviously, the culture medium of sulfate reducing bacteria used by oil field enterprises will underestimate the real number of hydrogen sulfide producing bacteria in the oil field environment, and further influence the water quality management and the research, development and decision of related hazard prevention and control technologies in the oil exploitation process.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a formula component of a culture medium which can effectively meet the growth and reproduction requirements of various hydrogen sulfide producing bacteria, and can realize the applications of enrichment culture, separation screening, quantity detection and the like of various hydrogen sulfide producing bacteria including sulfate reducing bacteria.
In order to achieve the purpose, the invention adopts the technical scheme that:
the culture medium for the hydrogen sulfide producing bacteria in the oil field comprises the following components in every 1000mL of the culture medium for the hydrogen sulfide producing bacteria: 924.0-961.7 mL of basic culture medium solution, 1.0-10.0 mL of carbon source solution, 1.0-10.0 mL of growth factor and nitrogen source solution, 1.0-10.0 mL of sulfur source solution, 0.1-1.0 mL of trace element solution, 0.1-1.0 mL of vitamin solution, 5.0-8.0 mL of reducing agent solution, 30.0-35.0 mL of pH buffer solution, 0.1-1.0 mL of ST solution and 0-20 g of agar.
The basic culture medium liquid isEach 1000mL of distilled water contains 0.05-30.0 g of NaCl and 0.05-5.0 g of MgCl2·6H2O、0.01～0.5g CaCl2·2H2O、0.05～10.0g Na2SO4、0.01～1.0g NH4Cl、0.05～3.0g KH2PO40.05-5.0 g KCl and 0-10.0 g MgSO4·7H2O, 0-10.0 g ferric ammonium citrate.
The carbon source solution contains 0.1-50 g of sodium lactate, 0.1-50 g of sodium acetate, 0.01-50 g of sodium formate, 0.01-50 g of sodium propionate, 0.01-50 g of sodium butyrate, 0-50 g of sodium succinate, 0-50 g of sodium malate and 0-50 g of sodium fumarate in each 100mL of distilled water.
The growth factor and nitrogen source solution contains 0.01-15.0 g of yeast extract and 0.01-15.0 g of peptone per 100mL of distilled water. The yeast extract contained in the growth factor and nitrogen source solution may be replaced by the equivalent trade name or popular name of "yeast extract", "yeast powder", "yeast extract powder", "yeast autolysate", "yeast extract powder", "yeast extract paste" or "yeast extract paste".
The sulfur source solution contains 0.5-50.0 g of Na in per 100mL of distilled water2SO3、0.5～80.0g Na2S2O3·5H2O and 0.5-5.0 g of elemental sulfur.
The trace element liquid contains 987-990 mL of distilled water, 10-13 mL of HCl (25%), and 10-5000 mg of FeSO in each 1000mL of trace element liquid4·7H2O、5～50mg H3BO3、10～200mg MnCl2·4H2O、10～300mg CoCl2·6H2O、10～100mg NiCl2·6H2O、5～50mg CuCl2·2H2O、10～500mg ZnSO4·7H2O and 10-200 mg of Na2MoO4·2H2O。
The vitamin solution contains 0.1-10 mg of aminobenzoic acid (4-aminobenzoic acid), 0.1-10 mg of biotin (D (+) -biotin), 0.1-20 mg of Nicotinic acid (Nicotinic acid) and 0.1-10 mg of calcium pantothenate in each 100mL of sodium phosphate buffer solution (10 mM; pH7.1)(Calcium D (+) -pantothenate), 0.1-10 mg vitamin B120.1-10 mg vitamin B1And 0.1-30 mg vitamin B6(Pyridoxine hydrochloride)。
The reducing agent solution is 0.1-0.5M of Na2S solution; the pH buffer was a 1.0M sodium bicarbonate solution.
The ST solution contains 0.1-1.0 g NaOH and 0.1-20 mg Na in each 1000mL of distilled water2SeO3·5H2O and 0.2-20 mg Na2WO4·2H2O。
An application of a culture medium for hydrogen sulfide producing bacteria in an oil field, wherein the culture medium is applied to enrichment culture, separation and screening, microorganism quantity measurement or monitoring of the hydrogen sulfide producing bacteria.
The invention has the beneficial effects that:
the invention can effectively avoid the defects of hydrogen sulfide producing bacteria quantity detection and monitoring value underestimation in the oil field environment water sample. The culture medium can be beneficial to separation and screening of hydrogen sulfide producing bacteria in samples in oil fields or other industries, can realize monitoring and detection of the quantity of the hydrogen sulfide producing bacteria, can further accurately monitor the dynamic change of the quantity of the hydrogen sulfide producing bacteria in the oil field development process, and is beneficial to guiding water quality management of oil field enterprises and improving the pertinence and effectiveness of harm prevention and control of the hydrogen sulfide producing bacteria.
Drawings
FIG. 1 is a graph showing the comparison between the number of hydrogen sulfide-producing bacteria in produced water of different oil fields in China measured by using the culture medium of the present invention and the number measured by a conventional method.
Detailed Description
The invention is further explained below with reference to the figures and examples.
The culture medium of the invention consists of basic culture medium solution, trace element solution, vitamin solution and the like, and also contains compound nutrient components such as organic carbon source, sulfur source and the like. Preparing each component solution of the culture medium according to a strict anaerobic method, and mixing according to a specific proportion to prepare the culture medium of the hydrogen sulfide producing bacteria. Under the anaerobic condition, a proper volume of an anaerobically collected oil field environmental water sample (such as produced water) is inoculated into the culture medium, and the anaerobic culture is carried out under the condition that the in-situ temperature of the environmental sample is the same, so that the growth and metabolism of various microorganism physiological groups with the capability of producing hydrogen sulfide in the sample can be efficiently promoted. By using the culture medium and combining other anaerobic microbiology methods, strain enrichment, separation and screening and microorganism quantity detection and monitoring of hydrogen sulfide producing bacteria in samples of oil field environments or other environmental sources can be effectively realized. The invention has important potential application value in the fields of oil exploitation, industrial water quality management and wastewater treatment, oil field microorganism acidification and control, microorganism corrosion monitoring and the like.
Example 1
The preparation method of the hydrogen sulfide producing bacteria culture medium suitable for medium salinity water comprises the following steps:
(1) and (4) preparing a basic culture medium solution. Accurately weighing 5.5g NaCl and 1.2g MgCl2·6H2O、0.05g CaCl2·2H2O、4.5g Na2SO4、0.2g NH4Cl、0.2g KH2PO4And 0.5g of KCl are sequentially dissolved into 1000mL of distilled water with the final volume, then the distilled water is heated and boiled, cooled under the protection of high-purity nitrogen, and transferred into an anaerobic bottle with a butyl rubber plug. Sterilizing at 121 deg.C for 20min with steam, and cooling;
(2) and (4) preparing a carbon source solution. 10.0g of sodium lactate, 10.0g of sodium acetate, 5.0g of sodium formate, 5.0g of sodium propionate, 5.0g of sodium butyrate, 5.0g of sodium succinate, 5.0g of sodium malate and 5.0g of sodium fumarate are accurately weighed and dissolved in 100mL of distilled water in turn. The anaerobic sterilization method of the carbon source solution is the same as that of the basic culture medium solution, and the sterilized solution is cooled for standby;
(3) preparing growth factor and nitrogen source solution. 5.0g of yeast extract and 5.0g of peptone were accurately weighed and dissolved in 100mL of distilled water. The anaerobic sterilization method of the growth factor and nitrogen source solution is the same as that of the (1), and the solution is cooled for standby after sterilization;
(4) and (4) preparing a sulfur source solution. Accurately weighing 12.0g of Na2SO3、24.0g Na2S2O3·5H2O dissolved in 100mL of distilled waterThen sterile filtration is carried out by using a sterile 0.22um filter membrane; then adding 0.5g of elemental sulfur powder subjected to ultraviolet sterilization for 30min for later use;
(5) and (4) preparing a trace element liquid. Accurately weighing 2.0g of FeSO4·7H2O、30mg H3BO3、100mg MnCl2·4H2O、190mg CoCl2·6H2O、24.0mg NiCl2·6H2O、5.0mg CuCl2·2H2O、100mg ZnSO4·7H2O and 36mg Na2MoO4·2H212.5mL of HCl (25%) was measured and dissolved in 987.5mL of distilled water. The anaerobic sterilization process of the trace element liquid is the same as that in the step (1); cooling the sterilized solution for later use;
(6) and (5) preparing a vitamin solution. Accurately weighing 4.0mg aminobenzoic acid (4-aminobenzoic acid), 1.0mg biotin (D (+) -biotin), 10.0mg Nicotinic acid (Nicotinic acid), 5mg Calcium pantothenate (Calcium D (+) -panthenate) and 5.0mg vitamin B1210mg of vitamin B115mg of vitamin B6(Pyridoxine hydrochloride) was dissolved in 100mL of a sodium phosphate buffer (10 mM; pH 7.1). The sterilization method of the vitamin solution is the same as that in the step (4);
(7) reducing agent solution-Na2And (5) preparing an S solution. Accurately weighing 4.8g of Na2S·9H2Dissolving O in 100mL of distilled water deoxidized by high-purity nitrogen in advance, sterilizing the solution by the same method as the step (4), and placing the solution in a sterile brown anaerobic bottle for later use;
(8) and (5) preparing an ST solution. Accurately weighing the mixture containing 0.4g of NaOH and 6.0mg of Na2SeO3·5H2O and 8mg Na2WO4·2H2O was dissolved in 1000mL of distilled water in this order, and the solution was sterilized in the same manner as in (1).
(9) In an anaerobic glove box, 1.0mL of a carbon source solution, 5.0mL of a growth factor and nitrogen source solution, 2.0mL of a sulfur source solution, 1.0mL of a trace element solution, 0.5mL of a vitamin solution, 5.0mL of a reducing agent solution, 33.0mL of a pH buffer solution, and 1.0mL of an ST solution were sequentially aspirated by a sterile syringe, added to 951.5mL of a basal medium solution, and the pH of the medium was adjusted to 7.5 with dilute hydrochloric acid or 0.2M NaOH solution which had been previously sterilized with steam. And then, aseptically subpackaging 9mL of the culture medium into sterile anaerobic tubes with the total volume of 18mL, and screwing and sealing by covering butyl rubber plugs and screw caps for later use.
Example 2
The preparation of the hydrogen sulfide producing bacteria culture medium suitable for the water with higher salinity: when the basic culture medium liquid of hydrogen sulfide-producing bacteria in example 1 was prepared, the amount of NaCl was increased to 12.0g/L, and the final pH of the culture medium was adjusted to 7.0 without changing the formulation of the remaining solutions and the mixing ratio of each solution.
Example 3
The preparation of the hydrogen sulfide producing bacteria culture medium suitable for offshore water salinity water quality: when the basic culture medium liquid of the hydrogen sulfide producing bacteria in the example 1 is prepared, the using amount of NaCl is increased to 20.0 g/L; sodium lactate in the carbon source solution is reduced to 5.0g/L, sodium acetate is reduced to 5g/L, and the use amounts of sodium formate, sodium propionate, sodium butyrate, sodium succinate, sodium malate and sodium fumarate are reduced to 1.0 g/L. The formulation components of the remaining solutions and the mixing ratio of each solution were unchanged, and the final pH of the medium was adjusted to 7.2.
Application example 1: detection of quantity of hydrogen sulfide producing bacteria in A oil field produced water in northeast China area
The culture medium of the embodiment 1 with a proper volume is taken and respectively inoculated to the produced water of 3 extraction wells of a certain oil field in the northeast of China, and the number of the hydrogen sulfide producing bacteria in the sample is determined by adopting a maximum possible counting method.
After the culture is carried out for 15 days at the culture temperature of 45 ℃, the result shows that the average number of the hydrogen sulfide producing bacteria in the produced water of the oil field measured by using the culture medium is 107On the order of one/ml and the commercial reagent bottle has a test result of 105One/ml. The number of hydrogen sulfide producing bacteria detected by the culture medium is significantly higher than that of the conventional common culture medium (see figure 1).
Application example 2: detection of quantity of hydrogen sulfide producing bacteria in B oil field produced water in northwest area of China
The culture medium of the embodiment 2 with a proper volume is taken and respectively inoculated to the produced water of 4 extraction wells of a certain oil field in the northwest area of China, and the number of the hydrogen sulfide producing bacteria in the sample is determined by adopting a maximum possible counting method.
After the culture is carried out for 15 days at the culture temperature of 37 ℃, the result shows that the average number of hydrogen sulfide producing bacteria in the produced water of the oil field measured by using the culture medium of the invention is 106On the order of one/ml and the commercial reagent bottle has a test result of 104One/ml. The number of hydrogen sulfide producing bacteria detected by the culture medium is significantly higher than that of the conventional common culture medium (see figure 1).
Application example 3: detection of quantity of hydrogen sulfide producing bacteria in C oil field produced water in Bohai Bay region of China
The culture medium of the embodiment 3 with a proper volume is taken and respectively inoculated to the produced water of 5 extraction wells of a certain oil field in the northeast of China, and the number of the hydrogen sulfide producing bacteria in the sample is determined by adopting a maximum possible counting method.
After the culture is carried out for 15 days at the culture temperature of 60 ℃, the result shows that the average number of hydrogen sulfide producing bacteria in the produced water of the oil field measured by using the culture medium of the invention is 105On the order of one/ml and the commercial reagent bottle has a test result of 102One/ml. The number of hydrogen sulfide producing bacteria detected by the culture medium is significantly higher than that of the conventional common culture medium (see figure 1).
Claims (2)
1. An oil field hydrogen sulfide producing bacteria culture medium is characterized in that: the culture medium of each 1000mL of hydrogen sulfide producing bacteria comprises the following components: 924.0-961.7 mL of basic culture medium solution, 1.0-10.0 mL of carbon source solution, 1.0-10.0 mL of growth factor and nitrogen source solution, 1.0-10.0 mL of sulfur source solution, 0.1-1.0 mL of trace element solution, 0.1-1.0 mL of vitamin solution, 5.0-8.0 mL of reducing agent solution, 30.0-35.0 mL of pH buffer solution, 0.1-1.0 mL of ST solution and 0-20 g of agar;
the basic culture medium solution contains 0.05-30.0 g NaCl and 0.05-5.0 g MgCl in each 1000mL of distilled water2·6H2O、0.01~0.5 g CaCl2·2H2O、0.05~10.0 g Na2SO4、0.01~1.0 g NH4Cl、0.05~3.0 g KH2PO4And 0.05 to 5.0g of KCl;
the culture medium is used for enrichment culture, separation screening, microorganism count determination or monitoring of hydrogen sulfide producing bacteria
The carbon source solution contains 0.1-50 g of sodium lactate, 0.1-50 g of sodium acetate, 0.01-50 g of sodium formate, 0.01-50 g of sodium propionate, 0.01-50 g of sodium butyrate, 0-50 g of sodium succinate, 0-50 g of sodium malate and 0-50 g of sodium fumarate in each 100mL of distilled water.
The growth factor and nitrogen source solution contains 0.01-15.0 g of yeast extract and 0.01-15.0 g of peptone per 100mL of distilled water.
The sulfur source solution contains 0.5-50.0 g of Na in per 100mL of distilled water2SO3、0.5~80.0 g Na2S2O3·5H2O and 0.5-5.0 g of elemental sulfur.
The trace element liquid contains 987-990 mL of distilled water, 10-13 mL of HCl (25%), and 10-5000 mg of FeSO in each 1000mL of trace element liquid4·7H2O、5~50 mg H3BO3、10~200 mg MnCl2·4H2O、10~300 mg CoCl2·6H2O、10~100 mg NiCl2·6H2O、5~50 mg CuCl2·2H2O、10~500 mg ZnSO4·7H2O and 10-200 mg of Na2MoO4·2H2O。
The vitamin solution is characterized in that each 100mL of sodium phosphate buffer solution (10 mM; pH7.1) contains 0.1-10 mg of aminobenzoic acid (4-aminobenzoic acid), 0.1-10 mg of biotin (D (+) -biotin), 0.1-20 mg of Nicotinic acid (Nicotinic acid), 0.1-10 mg of Calcium pantothenate (Calcium D (+) -panthenate) and 0.1-10 mg of vitamin B120.1-10 mg vitamin B1And 0.1-30 mg vitamin B6（Pyridoxine hydrochloride）。
The reducing agent solution is 0.1-0.5M of Na2S solution; the pH buffer is a 1.0M sodium bicarbonate solution;
the ST solution contains 0.1-1.0 g NaOH and 0.1-20 mg Na in each 1000mL of distilled water2SeO3·5H2O and 0.2-20 mg Na2WO4·2H2O。
2. The application of the culture medium of the oilfield hydrogen sulfide producing bacteria in claim 1 in enrichment culture, separation screening, microorganism counting or monitoring of the hydrogen sulfide producing bacteria.
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