Culture medium for screening and purifying reducing bacteria with conductive pili

Abstract

The invention discloses a culture medium for screening and purifying reducing bacteria with conductive pili. The culture medium comprises 0.05 to 2.5 g / L of peptone, 0.05 to 2.5 g / L of yeast extract, 0.02 to 1 g / L of sodium chloride, 0.3 to 15 g / L of phosphate, 0.5 to 25 g / L of sodium acetate, 0 to 5 g / L of sodium citrate, 0.2 to 10 g / L of sodium lactate, 0.02 to 1 g / L of calcium chloride, 0.1 to 5 g / L of sodium bicarbonate, 0.02 to 1 g / L of L-cysteine and 0.01 to 20 g / L of electron acceptor. A solvent in the culture medium is water. The culture medium disclosed by the invention can promote efficient proliferation of the reducing bacteria with conductive pili, and the reducing bacteria with conductive pili can be visually and accurately screened and identified according to color change or precipitation formation.

Description

Technical Field

[0001] The invention belongs to the technical field of bacterial culture, screening and separation, and in particular relates to a culture medium for screening and purifying reducing bacteria with conductive pili. Background Art

[0002] Since their discovery, reductive bacteria have attracted widespread attention from scientific research and industry due to their unique metabolic characteristics and wide application potential in environmental remediation, industrial production, and bioenergy development.

[0003] Reductive bacteria with conductive pili are a type of reductive bacteria, widely distributed in water sediments, soil and various underground anaerobic environments. In the process of culturing these bacteria, the selection and preparation of culture media are particularly important. Most of the widely used ones are NB culture media or NBAF culture media with some special ingredients added. For example, electron acceptors need to be added to meet the metabolic needs of bacterial growth to release electrons. Fumaric acid (fumaric acid) is often used as electron acceptor. With the deepening of research, researchers have a deeper understanding of the nutritional needs of reductive bacteria and found that these traditional culture media cannot meet the growth needs of specific reductive bacteria in some cases.

[0004] At present, the main shortcomings and disadvantages of reducing culture medium formulas are their lack of specificity and selectivity. An ideal reducing culture medium should be able to provide an environment that allows reducing bacteria to grow preferentially while inhibiting or significantly slowing the growth of other types of bacteria. However, existing formulas cannot meet this standard. They either cannot effectively promote the growth of reducing bacteria, or they are suitable for reducing bacteria, but are also suitable for the growth of some fermentative bacteria. Therefore, during the culture process, the culture medium can only play the role of enrichment culture, without the role of selection and screening, and cannot play an indicative role. This not only increases the complexity of the experiment, but may also lead to misjudgment of the results. For example, the growth of fermentative bacteria during enrichment culture may consume key nutrients in the culture medium, inhibit the growth and metabolic activity of reducing bacteria, or be easily masked by other fast-growing bacteria during the separation process, thereby affecting the accuracy and repeatability of the experiment.

[0005] After separation and purification, the determination of the bacterial species can only be judged by combining morphology and gene sequencing because it is impossible to observe macroscopically. Morphological identification relies on observation under a microscope, but this method has the problems of strong subjectivity and large errors. Although gene sequencing provides a more accurate means of identification, it is costly, time-consuming, and requires professional equipment and technicians. The early cultivation stage lacks clear targeting, and the identification of bacterial species is cumbersome and time-consuming. In addition, whether the target strain can be obtained after a long period of screening and purification depends largely on accidental factors. These problems lead to the inefficiency and long cycle of the process of isolating and purifying the target strain, which seriously restricts the subsequent research progress and application development. Summary of the invention

[0006] In view of the problem that the existing reducing bacteria culture medium formula lacks specificity and selectivity, the present invention provides a culture medium for screening and purifying reducing bacteria with conductive pili. The culture medium of the present invention can promote the efficient proliferation of reducing bacteria with conductive pili, and can intuitively and accurately screen and identify reducing bacteria with conductive pili according to color changes or precipitation formation, greatly improving the screening efficiency and accuracy, and showing good application prospects.

[0007] The present invention solves the above technical problems through the following technical solutions:

[0008] The invention provides a culture medium, comprising 0.05-2.5 g / L peptone, 0.05-2.5 g / L yeast extract, 0.02-1 g / L sodium chloride, 0.3-15 g / L phosphate, 0.5-25 g / L sodium acetate, 0-5 g / L sodium citrate, 0.2-10 g / L sodium lactate, 0.02-1 g / L calcium chloride, 0.1-5 g / L sodium bicarbonate, 0.02-1 g / L L-cysteine ​​and 0.01-20 g / L electron acceptor; the solvent in the culture medium is water; wherein g / L represents the ratio of the mass of each component to the total volume of the culture medium;

[0009] When the electron acceptor is manganese dioxide, or iron oxide and / or a sulfur-containing reagent; the total amount of the phosphate, sodium chloride, sodium citrate, sodium lactate, calcium chloride and sodium bicarbonate does not exceed 15 g / L;

[0010] When the electron acceptor is dichromate, the content of the electron acceptor is 0.01-5 g / L; the total amount of the phosphate, sodium chloride, sodium citrate, sodium lactate, calcium chloride, sodium bicarbonate and dichromate does not exceed 15 g / L.

[0011] In a certain embodiment, the culture medium further comprises 1-50 ml / L Wolfe's Vitamin Solution, preferably 1-10 ml / L Wolfe's Vitamin Solution; ml / L represents the ratio of the volume of Wolfe's Vitamin Solution to the total volume of the culture medium.

[0012] In a certain embodiment, the culture medium further comprises 1-50 ml / L Modified Wolfe's Minerals, preferably 1-10 ml / L Modified Wolfe's Minerals; ml / L represents the ratio of the volume of Modified Wolfe's Minerals to the total volume of the culture medium.

[0013] In one embodiment, the culture medium further comprises 1.5-30 g / L agar powder, such as 15-20 g / L agar powder.

[0014] In one embodiment, the pH of the culture medium is 6.5-8.

[0015] In a certain embodiment, the content of peptone is 0.05-1.3 g / L; for example, 0.5 g / L, 0.55 g / L or 1.25 g / L.

[0016] In a certain embodiment, the content of the yeast extract is 0.05-1.3 g / L; for example, 0.5 g / L, 0.55 g / L or 1.25 g / L.

[0017] In a certain embodiment, the phosphate is 0.1-5 g / L potassium dihydrogen phosphate, 0.1-5 g / L sodium dihydrogen phosphate and 0.1-5 g / L ammonium dihydrogen phosphate.

[0018] In a certain embodiment, the phosphate is 1 g / L potassium dihydrogen phosphate, 1 g / L sodium dihydrogen phosphate and 1 g / L ammonium dihydrogen phosphate.

[0019] In a certain embodiment, the content of L-cysteine ​​is 0.2 g / L.

[0020] In one embodiment, the water is distilled water or deionized water.

[0021] In a certain embodiment, when the electron acceptor is dichromate, the dichromate is potassium dichromate, for example, 0.1-5 g / L potassium dichromate.

[0022] In one embodiment, when the electron acceptor is dichromate, the pH of the culture medium is 7-8.

[0023] In a certain embodiment, when the electron acceptor is manganese dioxide, the content of the manganese dioxide is 2.5 g / L; the manganese dioxide is preferably prepared by the following method: grinding the manganese dioxide powder using a ball mill and sieving it through a 600 mesh screen.

[0024] In a certain embodiment, the iron oxide is low-crystallinity iron oxide; the low-crystallinity iron oxide is preferably prepared by the following method: grinding the iron oxide powder using a ball mill and sieving it through a 600-mesh sieve.

[0025] In a certain embodiment, the sulfur-containing reagent is sulfate or sulfur powder, preferably magnesium sulfate, potassium sulfate or sulfur powder.

[0026] In a certain embodiment, when the electron acceptor is iron oxide and / or a sulfur-containing reagent, the culture medium may further include ferrous ions, and the ferrous ions are preferably ferrous chloride; for example, 0.05-2.5 g / L ferrous chloride; for example, 0.5 g / L ferrous chloride;

[0027] In a certain embodiment, the electron acceptor is preferably a combination of iron oxide and magnesium sulfate, or a combination of iron oxide and sulfur powder.

[0028] In a certain embodiment, when the electron acceptor is a combination of iron oxide and a sulfur-containing reagent, the content of the electron acceptor is 0.15~7.5 g / L, 0.3~15 g / L or 0.4~20 g / L; wherein the content of the iron oxide is 0.05~10 g / L, for example, 0.5 g / L or 2 g / L; the content of the sulfur-containing reagent is 0.1~10 g / L, preferably 0.5~2 g / L; for example, 1 g / L or 2 g / L.

[0029] In a certain embodiment, when the electron acceptor is preferably a combination of iron oxide and magnesium sulfate, the content of the iron oxide is 0.5 g / L or 2 g / L; the content of the magnesium sulfate is 0.5-1 g / L.

[0030] In a certain embodiment, when the electron acceptor is preferably a combination of iron oxide and sulfur powder, the content of the iron oxide is 2 g / L; the content of the sulfur powder is 2 g / L.

[0031] In a certain embodiment, when the electron acceptor is preferably a combination of iron oxide and sulfur powder, the culture medium further includes 0.1-5 g / L magnesium chloride, for example 1 g / L magnesium chloride.

[0032] In a certain embodiment, the culture medium includes 0.05~1.25g / L peptone, 0.05~1.25g / L yeast extract, 0.5~1g / L potassium dihydrogen phosphate, 0.5~1g / L sodium dihydrogen phosphate, 0.2g / L sodium chloride, 0.5~1g / L ammonium dihydrogen phosphate, 0.2g / L calcium chloride, 0.5~1g / L sodium bicarbonate, 5~12.5g / L sodium acetate, 0.5~2g / L sodium lactate, 0.5~1g / L sodium citrate, 0.2g / L L-cysteine, 0.5~2g / L sulfur-containing reagent, 0.5~2g / L iron oxide powder, 1~10ml / L Wolfe's Vitamin Solution, 1~10ml / L Modified Wolfe's Minerals and / or 15-20 g / L agar powder, and / or 0.5 g / L ferrous chloride; the solvent in the culture medium is water.

[0033] In a certain embodiment, the culture medium is any of the following formulas:

[0034] Formulation 1: 0.5 g / L peptone, 0.5 g / L yeast extract, 1 g / L potassium dihydrogen phosphate, 1 g / L sodium dihydrogen phosphate, 0.2 g / L sodium chloride, 1 g / L ammonium dihydrogen phosphate, 0.2 g / L calcium chloride, 1 g / L sodium bicarbonate, 5 g / L sodium acetate, 2 g / L sodium lactate, 1 g / L sodium citrate, 0.2 g / L L-cysteine, 1 g / L magnesium sulfate, 2 g / L low crystallinity iron oxide powder, 10 ml / L Wolfe's Vitamin Solution and 10 ml / L Modified Wolfe's Minerals; the solvent in the culture medium is water.

[0035] Formulation 2: 0.55~1.25g / L peptone, 0.55~1.25g / L yeast extract, 0.5~1g / L potassium dihydrogen phosphate, 0.5~1g / L sodium dihydrogen phosphate, 0.2g / L sodium chloride, 0.5~1g / L ammonium dihydrogen phosphate, 0.2g / L calcium chloride, 0.5~1g / L sodium bicarbonate, 5.5~12.5g / L sodium acetate, 0.5~2g / L sodium lactate, 0.5~1g / L sodium citrate, 0.2g / L L-cysteine, 0.5~1g / L magnesium sulfate, 2g / L low crystallinity iron oxide powder, 1~10ml / L Wolfe's Vitamin Solution and 1~10ml / L Modified Wolfe's Minerals; the solvent in the culture medium is water.

[0036] Formulation 3: 0.5 g / L peptone, 0.5 g / L yeast extract, 1 g / L potassium dihydrogen phosphate, 1 g / L sodium dihydrogen phosphate, 0.2 g / L sodium chloride, 1 g / L ammonium dihydrogen phosphate, 0.2 g / L calcium chloride, 1 g / L sodium bicarbonate, 5 g / L sodium acetate, 2 g / L sodium lactate, 1 g / L sodium citrate, 0.2 g / L cysteine, 1 g / L magnesium sulfate, 0.5 g / L low crystallinity iron oxide powder, 15-20 g / L agar powder, 10 ml / L Wolfe's Vitamin Solution and 10 ml / L Modified Wolfe's Minerals; the solvent in the culture medium is water.

[0037] Formulation 4: 0.05 g / L peptone, 0.05 g / L yeast extract, 1 g / L potassium dihydrogen phosphate, 1 g / L sodium dihydrogen phosphate, 1 g / L magnesium chloride, 0.2 g / L sodium chloride, 1 g / L ammonium dihydrogen phosphate, 0.2 g / L calcium chloride, 1 g / L sodium bicarbonate, 5 g / L sodium acetate, 2 g / L sodium lactate, 1 g / L sodium citrate, 0.2 g / L L-cysteine, 2 g / L sulfur powder, 2 g / L low crystallinity iron oxide powder, 10 ml / L Wolfe's Vitamin Solution and 10 ml / L Modified Wolfe's Minerals; the solvent in the culture medium is water.

[0038] Formulation 5: 0.5 g / L peptone, 0.5 g / L yeast extract, 1 g / L potassium dihydrogen phosphate, 1 g / L sodium dihydrogen phosphate, 0.2 g / L sodium chloride, 1 g / L ammonium dihydrogen phosphate, 0.2 g / L calcium chloride, 1 g / L sodium bicarbonate, 5 g / L sodium acetate, 2 g / L sodium lactate, 1 g / L sodium citrate, 0.2 g / L L-cysteine, 1 g / L magnesium sulfate, 2 g / L low crystallinity iron oxide powder, 0.5 g / L ferrous chloride, 10 ml / L Wolfe's Vitamin Solution and 10 ml / L Modified Wolfe's Minerals; the solvent in the culture medium is water.

[0039] The present invention provides a method for preparing a culture medium, comprising the following steps:

[0040] Deoxygenating the mixture A, sterilizing it under high pressure, cooling it, and then mixing it with the mixture B to obtain the culture medium;

[0041] The mixture A comprises 0.05-2.5 g / L peptone, 0.05-2.5 g / L yeast extract, 0.02-1 g / L sodium chloride, 0.3-15 g / L phosphate, 0.5-25 g / L sodium acetate, 0.02-1 g / L calcium chloride and 0.01-20 g / L electron acceptor;

[0042] The mixture B comprises 0-5 g / L sodium citrate, 0.2-10 g / L sodium lactate, 0.1-5 g / L sodium bicarbonate and 0.02-1 g / L L-cysteine; the solvent in the culture medium is water; wherein g / L represents the ratio of the mass of each component to the total volume of the culture medium;

[0043] When the electron acceptor is manganese dioxide, or iron oxide and / or a sulfur-containing reagent; the total amount of phosphate, sodium chloride, sodium citrate, sodium lactate, calcium chloride and sodium bicarbonate in the mixture A and the mixture B does not exceed 15 g / L;

[0044] When the electron acceptor is dichromate, the content of the electron acceptor is 0.01-5 g / L; the total amount of phosphate, sodium chloride, sodium citrate, sodium lactate, calcium chloride, sodium bicarbonate, and dichromate in the mixture A and mixture B does not exceed 15 g / L;

[0045] In a certain embodiment, the mixture B further comprises 1-50 ml / L Wolfe's Vitamin Solution, preferably 1-10 ml / L Wolfe's Vitamin Solution; ml / L represents the ratio of the volume of Wolfe's Vitamin Solution to the total volume of the culture medium.

[0046] In a certain embodiment, the mixture B further comprises 1-50 ml / L Modified Wolfe's Minerals, preferably 1-10 ml / L Modified Wolfe's Minerals; ml / L represents the ratio of the volume of Modified Wolfe's Minerals to the total volume of the culture medium.

[0047] In one embodiment, the mixed solution A further comprises 1.5-30 g / L agar powder; for example, 15-20 g / L agar powder.

[0048] In one embodiment, the pH of the culture medium is 6.5-8.

[0049] In a certain embodiment, the content of peptone is 0.05-1.3 g / L; for example, 0.5 g / L, 0.55 g / L or 1.25 g / L.

[0050] In a certain embodiment, the content of the yeast extract is 0.05-1.3 g / L; for example, 0.5 g / L, 0.55 g / L or 1.25 g / L.

[0051] In a certain embodiment, the phosphate is 0.1-5 g / L potassium dihydrogen phosphate, 0.1-5 g / L sodium dihydrogen phosphate and 0.1-5 g / L ammonium dihydrogen phosphate.

[0052] In a certain embodiment, the phosphate is 1 g / L potassium dihydrogen phosphate, 1 g / L sodium dihydrogen phosphate and 1 g / L ammonium dihydrogen phosphate.

[0053] In a certain embodiment, the content of L-cysteine ​​is 0.2 g / L.

[0054] In one embodiment, the water is distilled water or deionized water.

[0055] In a certain embodiment, when the electron acceptor is dichromate, the dichromate is potassium dichromate, for example, 0.1-5 g / L potassium dichromate.

[0056] In one embodiment, when the electron acceptor is dichromate, the pH of the culture medium is 7-8.

[0057] In a certain embodiment, when the electron acceptor is manganese dioxide, the content of the manganese dioxide is 2.5 g / L; the manganese dioxide is preferably prepared by the following method: grinding the manganese dioxide powder using a ball mill and sieving it through a 600 mesh screen.

[0058] In a certain embodiment, the iron oxide is low-crystallinity iron oxide; the low-crystallinity iron oxide is preferably prepared by the following method: grinding the iron oxide powder using a ball mill and sieving it through a 600-mesh sieve.

[0059] In a certain embodiment, the sulfur-containing reagent is sulfate or sulfur powder, preferably magnesium sulfate, potassium sulfate or sulfur powder.

[0060] In a certain embodiment, when the electron acceptor is iron oxide and / or a sulfur-containing reagent, the culture medium may further include ferrous ions, and the ferrous ions are preferably ferrous chloride; for example, 0.05-2.5 g / L ferrous chloride; for example, 0.5 g / L ferrous chloride.

[0061] In a certain embodiment, the electron acceptor is preferably a combination of iron oxide and magnesium sulfate, or a combination of iron oxide and sulfur powder.

[0062] In a certain embodiment, when the electron acceptor is a combination of iron oxide and a sulfur-containing reagent, the content of the electron acceptor is 0.15~7.5g / L, 0.3~15g / L or 0.4~20g / L; wherein the content of the iron oxide is 0.05~10g / L, for example, 0.5g / L or 2g / L; the content of the sulfur-containing reagent is 0.1~10g / L, preferably 0.5~2g / L; for example, 1g / L or 2g / L.

[0063] In a certain embodiment, when the electron acceptor is preferably a combination of iron oxide and magnesium sulfate, the content of the iron oxide is 0.5 g / L or 2 g / L; the content of the magnesium sulfate is 0.5-1 g / L.

[0064] In a certain embodiment, when the electron acceptor is preferably a combination of iron oxide and sulfur powder, the content of the iron oxide is 2 g / L; the content of the sulfur powder is 2 g / L.

[0065] In a certain embodiment, when the electron acceptor is preferably a combination of iron oxide and sulfur powder, the culture medium further includes 0.1-5 g / L magnesium chloride, for example 1 g / L magnesium chloride.

[0066] In one embodiment, the culture medium is deoxygenated by passing a mixed gas of an inert gas and carbon dioxide, wherein the inert gas is preferably nitrogen.

[0067] In a certain embodiment, in the mixed gas, the volume ratio of the inert gas to the carbon dioxide is (60-90):(40-10), for example, 80:20.

[0068] In one embodiment, the high pressure sterilization temperature is 110-135°C, for example 121°C.

[0069] In a certain embodiment, the high pressure sterilization time is 10 to 30 minutes, for example, 15 minutes.

[0070] In one embodiment, the cooling temperature is 10-50°C, such as 25°C or 45°C.

[0071] The present invention provides a culture medium, which is prepared by the above preparation method.

[0072] The present invention also provides a use of the culture medium as described above in screening and purifying reducing bacteria having conductive pili.

[0073] In a certain embodiment, the reducing bacteria of the conductive pili are Geobacter reductus, sulfate-reducing bacteria or metal-reducing bacteria.

[0074] The culture medium of the present invention is used for conventional purposes in the art, such as in strain enrichment, primary screening or separation and purification processes.

[0075] The source of the inoculated strain in the culture medium of the present invention may be water sediment or underground soil, such as drainage ditch mud, lake or pond mud or deep humus soil (below 20 cm).

[0076] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0077] The reagents and raw materials used in the present invention are commercially available.

[0078] The positive and progressive effect of the present invention is that the culture medium of the present invention uses iron oxide and / or sulfur-containing reagents, or dichromates, or manganese dioxide as electron acceptors, and organic acid salts as carbon sources, which effectively overcomes the two inherent defects of traditional culture media, namely, lack of targeting and indicativeness. While promoting the growth of reducing bacteria with conductive pili, it inhibits and slows down the growth of other types of bacteria, thereby greatly reducing the interference of foreign bacteria in the culture process. According to color changes or precipitation formation, the present invention can accurately indicate the potential screening situation of the target strain, thereby greatly eliminating the interference of non-target strains in the identification process during the strain screening process, and achieving a significant simplification of the screening process. Not only does it reduce the complexity and pressure of the later identification work, but it also greatly reduces the workload of screening and purification of the target strain, thereby significantly improving the overall work efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 For the cultivation process.

[0080] Figure 2 For the purification process. DETAILED DESCRIPTION

[0081] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0082] Wolfe's Vitamin Solution: A Wolfe's Vitamin Solution preparation with double the concentration produced by Huzhen Biotechnology or Zeye Biotechnology.

[0083] Modified Wolfe's Minerals: A Modified Wolfe's Minerals preparation with double the concentration of that produced by Huzhen Biotechnology or Zeye Biotechnology.

[0084] Example 1

[0085] Liquid culture medium formula 1 is as follows (per liter): 1g potassium dihydrogen phosphate, 1g sodium dihydrogen phosphate, 1g magnesium sulfate, 0.2g sodium chloride, 5g sodium acetate, 0.5g yeast extract, 0.5g peptone (the amount of sodium acetate, yeast extract and peptone can be 1.1-2.5 times the formula amount), 1g ammonium dihydrogen phosphate, 1g sodium citrate, 2g low-crystallinity iron oxide powder (ground using a ball mill and sieved through 600 mesh), 0.2g calcium chloride, 1g sodium bicarbonate, 2g sodium lactate, 0.2g L-cysteine, 10ml Wolfe's Vitamin Solution, and 10ml Modified Wolfe's Minerals.

[0086] According to liquid culture medium formula 1, weigh (potassium dihydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, sodium chloride, ammonium dihydrogen phosphate, low-crystallinity iron oxide, calcium chloride), as well as sodium acetate, yeast extract and peptone and dissolve them in 980 ml of distilled water or deionized water (iron oxide powder is insoluble in water and exists in a precipitated state), pass N2-CO2 (80:20) to remove oxygen from the culture medium, sterilize at 121°C for 15 minutes, and then cool to below 35°C, weigh sodium citrate, sodium bicarbonate, sodium lactate, L-cysteine, Wolfe's Vitamin Solution, and Modified Wolfe's Minerals, add them to dissolve, shake well and divide into portions as enrichment culture medium for later use.

[0087] Take 20g of the drainage ditch sludge, add 250mL of sterile water, stir and shake for 5 minutes, take 5ml of the supernatant and add it to the enrichment medium for enrichment. Under anaerobic, 30℃~37℃, and shaking culture, an obvious rotten egg smell will appear in two to three days, indicating that the reducing bacteria have multiplied in large numbers.

[0088] Example 2

[0089] According to liquid culture medium formula 1, weigh (potassium dihydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, sodium chloride, ammonium dihydrogen phosphate, low-crystallinity iron oxide, calcium chloride), as well as sodium acetate, yeast extract, and peptone and dissolve them in 980 ml of distilled water or deionized water (iron oxide powder is insoluble in water and exists in a precipitated state), pass N2-CO2 (80:20) to remove oxygen from the culture medium, sterilize at 121°C for 15 minutes, and then cool to below 35°C, weigh sodium citrate, sodium bicarbonate, sodium lactate, L-cysteine, Wolfe's Vitamin Solution, and Modified Wolfe's Minerals, add them to dissolve, shake well, and divide into portions as primary screening culture medium for later use.

[0090] During the initial screening process, dilute the enriched culture solution 1000 times, take 1-2 ml and add it to the initial screening culture medium, and culture it in a shaking table under anaerobic conditions at 30℃~37℃. When specific black precipitate, black foam, and black suspended matter appear, it can be judged that bacteria with conductive pili reduction have been screened out with a high probability (the cultivation process is as follows Figure 1 as shown).

[0091] During the purification process, in order to minimize the interference of foreign bacteria, the initial screening operation can be further repeated (refer to the purification process Figure 2 ). The strains obtained after preliminary purification were collected and sequenced using high-throughput sequencing technology. The results are as follows: 18% desulfovibrio was found in the sample.

[0092]

[0093] Example 3

[0094] This culture medium can also be used as a solid culture medium.

[0095] Solid culture medium formula 1 is as follows (per liter): potassium dihydrogen phosphate 1g, sodium dihydrogen phosphate 1g, magnesium sulfate 1g, sodium chloride 0.2g, sodium acetate 5g, yeast extract 0.5g, peptone 0.5g, ammonium dihydrogen phosphate 1g, sodium citrate 1g, low-crystallinity iron oxide powder (ground by ball mill and sieved through 600 mesh) 0.5g, calcium chloride 0.2g, sodium bicarbonate 1g, sodium lactate 2g, L-cysteine ​​0.2g, Wolfe's Vitamin Solution 10ml, Modified Wolfe's Minerals 10ml, and agar powder 15-20g.

[0096] According to the formula, weigh (potassium dihydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, sodium chloride, sodium acetate, yeast extract, peptone, ammonium dihydrogen phosphate, low-crystallization iron oxide, calcium chloride, agar powder) and dissolve in 980 ml of distilled water or deionized water (iron oxide powder is insoluble in water and exists in a precipitated state), pass N2-CO2 (80:20) to remove oxygen in the culture medium, sterilize at 121℃ for 15 minutes, and when cooled to 45℃, weigh sodium citrate, sodium bicarbonate, sodium lactate, L-cysteine, Wolfe's Vitamin Solution, and Modified Wolfe's Minerals and add them to dissolve, shake well, pour the culture medium into the sterilized culture dish, cover the dish, wait for the culture medium to cool and solidify, and then invert the plate for later use. Dilute the purified bacterial solution 100 times, spread it on the plate, and culture it under anaerobic conditions at 30℃~37℃. Black colonies will grow in 2~5 days.

[0097] Example 4

[0098] Adjustment of the content of formula ingredients: The content of each ingredient in the formula can be adjusted, and each ingredient can be adjusted by 0.1~5 times. The comprehensive salinity is recommended to be less than 15g / L. If the salinity is too high, it will significantly inhibit the growth of bacteria and prolong the separation and screening process.

[0099] Example 5

[0100] Formula adjustment to enhance purification effect: The culture medium formula is as follows (per liter): 1g potassium dihydrogen phosphate, 1g sodium dihydrogen phosphate, 1g magnesium chloride, 0.2g sodium chloride, 5g sodium acetate, 0.05g yeast extract, 0.05g peptone, 1g ammonium dihydrogen phosphate, 1g sodium citrate, 2g low-crystallinity iron oxide powder, 2g sulfur powder, 0.2g calcium chloride, 1g sodium bicarbonate, 2g sodium lactate, 0.2g L-cysteine, 10ml Wolfe's Vitamin Solution, 10ml Modified Wolfe's Minerals.

[0101] Example 6

[0102] Adjustment of the rapid screening formula: To obtain screening results more quickly, Formula 1 can add 0.5g / L of ferrous chloride (other forms of ferrous ions can also achieve this effect). When there are more ferrous ions in the solution, since sulfate ions dissolved in water can be reduced and utilized in more forms, the metabolic efficiency of bacteria on sulfate is significantly higher than that of iron oxide. When sulfate is reduced to hydrogen sulfide, it will immediately form a black precipitate with ferrous ions, which can provide indication results more quickly.

[0103] Example 7

[0104] Formula indicator adjustment: Other metal ions with obvious color changes can be used as indicators. For example, iron oxide powder and / or sulfur-containing reagents can be replaced with dichromate or manganese dioxide, but the initial salinity should be controlled to less than 15g / L to prevent failure of initial development.

[0105] When the electron acceptor is potassium dichromate, the dosage should be controlled at 0.1-5 g / L, and the pH value should be controlled at 7-8. The color of the culture medium changes from orange-red to green.

[0106] When the electron acceptor is manganese dioxide, the dosage is 2.5 g / L (grind the manganese dioxide powder with a ball mill and sieve through 600 mesh). During the culture process, the culture medium will gradually fade from a black suspension state to light gray, or colorless or light pink.

Claims

1. A culture medium, characterized in that The culture medium comprises 0.05-2.5 g / L peptone, 0.05-2.5 g / L yeast extract, 0.02-1 g / L sodium chloride, 0.3-15 g / L phosphate, 0.5-25 g / L sodium acetate, 0-5 g / L sodium citrate, 0.2-10 g / L sodium lactate, 0.02-1 g / L calcium chloride, 0.1-5 g / L sodium bicarbonate, 0.02-1 g / L L-cysteine ​​and 0.01-20 g / L electron acceptor; the solvent in the culture medium is water; Where g / L represents the ratio of the mass of each component to the total volume of the culture medium; When the electron acceptor is manganese dioxide, or iron oxide and / or a sulfur-containing reagent; the total amount of the phosphate, sodium chloride, sodium citrate, sodium lactate, calcium chloride and sodium bicarbonate does not exceed 15 g / L; When the electron acceptor is dichromate, the content of the electron acceptor is 0.01-5 g / L; the total amount of the phosphate, sodium chloride, sodium citrate, sodium lactate, calcium chloride, sodium bicarbonate and dichromate does not exceed 15 g / L.

2. The culture medium according to claim 1, characterized in that It meets one or more of the following conditions: (1) The culture medium also includes 1-50 ml / L Wolfe's Vitamin Solution; (2) The culture medium further comprises 1-50 ml / L Modified Wolfe's Minerals; (3) The culture medium also includes 1.5 to 30 g / L agar powder; (4) The pH of the culture medium is 6.5-8; (5) The content of peptone is 0.05-1.3 g / L; (6) The yeast extract has a content of 0.05 to 1.3 g / L; (7) The phosphate is 0.1-5 g / L potassium dihydrogen phosphate, 0.1-5 g / L sodium dihydrogen phosphate and 0.1-5 g / L ammonium dihydrogen phosphate; (8) The content of L-cysteine ​​is 0.2 g / L; (9) The water is distilled water or deionized water; (10) When the electron acceptor is dichromate, the dichromate is potassium dichromate; (11) When the electron acceptor is manganese dioxide, the content of manganese dioxide is 2.5 g / L; (12) The iron oxide is low crystallinity iron oxide; (13) The sulfur-containing reagent is sulfate or sulfur powder; preferably magnesium sulfate, potassium sulfate or sulfur powder; (14) When the electron acceptor is iron oxide and / or a sulfur-containing reagent, the culture medium further comprises ferrous ions; the ferrous ions are preferably ferrous chloride; for example, 0.05 to 2.5 g / L ferrous chloride; for example, 0.5 g / L ferrous chloride.

3. The culture medium according to claim 2, characterized in that It meets one or more of the following conditions: (1) The culture medium further comprises 1-10 ml / L Wolfe's Vitamin Solution; (2) The culture medium further comprises 1-10 ml / L Modified Wolfe's Minerals; (3) The culture medium also includes 15-20 g / L agar powder; (4) The content of the peptone is 0.5 g / L, 0.55 g / L or 1.25 g / L; (5) The yeast extract content is 0.5 g / L, 0.55 g / L or 1.25 g / L; (6) The phosphate is 1 g / L potassium dihydrogen phosphate, 1 g / L sodium dihydrogen phosphate and 1 g / L ammonium dihydrogen phosphate; (7) When the electron acceptor is dichromate, the dichromate is 0.1-5 g / L potassium dichromate; (8) When the electron acceptor is dichromate, the pH of the culture medium is 7-8; (9) When the electron acceptor is manganese dioxide, the manganese dioxide is prepared by the following method: grinding manganese dioxide powder using a ball mill and sieving through a 600 mesh screen; (10) The low crystallinity iron oxide is prepared by the following method: grinding the iron oxide powder using a ball mill and sieving it through a 600 mesh screen; (11) When the electron acceptor is a combination of iron oxide and a sulfur-containing reagent, the content of the electron acceptor is 0.15-7.5 g / L, 0.3-15 g / L or 0.4-20 g / L; wherein the content of the iron oxide is 0.05-10 g / L, such as 0.5 g / L or 2 g / L; the content of the sulfur-containing reagent is 0.1-10 g / L, preferably 0.5-2 g / L; such as 1 g / L or 2 g / L; (12) When the electron acceptor is a combination of iron oxide and sulfur powder, the culture medium further includes 0.1-5 g / L magnesium chloride, for example, 1 g / L magnesium chloride.

4. The culture medium according to claim 1, characterized in that The culture medium comprises 0.05-1.25 g / L peptone, 0.05-1.25 g / L yeast extract, 0.5-1 g / L potassium dihydrogen phosphate, 0.5-1 g / L sodium dihydrogen phosphate, 0.2 g / L sodium chloride, 0.5-1 g / L ammonium dihydrogen phosphate, 0.2 g / L calcium chloride, 0.5-1 g / L sodium bicarbonate, 5-12.5 g / L sodium acetate, 0.5-2 g / L sodium lactate, 0.5-1 g / L sodium citrate, 0.2 g / L L-cysteine, 0.5-2 g / L sulfur-containing reagent, 0.5-2 g / L iron oxide powder, 1-10 ml / L Wolfe's Vitamin Solution, 1-10 ml / L Modified Wolfe's Minerals and / or 15-20 g / L agar powder, and / or 0.5 g / L ferrous chloride; and the solvent in the culture medium is water.

5. The culture medium according to claim 4, characterized in that The culture medium is any of the following formulas: Formulation 1: 0.5 g / L peptone, 0.5 g / L yeast extract, 1 g / L potassium dihydrogen phosphate, 1 g / L sodium dihydrogen phosphate, 0.2 g / L sodium chloride, 1 g / L ammonium dihydrogen phosphate, 0.2 g / L calcium chloride, 1 g / L sodium bicarbonate, 5 g / L sodium acetate, 2 g / L sodium lactate, 1 g / L sodium citrate, 0.2 g / L L-cysteine, 1 g / L magnesium sulfate, 2 g / L low crystallinity iron oxide powder, 10 ml / L Wolfe's Vitamin Solution and 10 ml / L Modified Wolfe's Minerals; the solvent in the culture medium is water; Formula 2: 0.55~1.25g / L peptone, 0.55~1.25g / L yeast extract, 0.5~1g / L potassium dihydrogen phosphate, 0.5~1g / L sodium dihydrogen phosphate, 0.2g / L sodium chloride, 0.5~1g / L ammonium dihydrogen phosphate, 0.2g / L calcium chloride, 0.5~1g / L sodium bicarbonate, 5.5~12.5g / L sodium acetate, 0.5~2g / L sodium lactate, 0.5~1g / L sodium citrate, 0.2g / L L-cysteine, 0.5~1g / L magnesium sulfate, 2g / L low crystallinity iron oxide powder, 1~10ml / L Wolfe's Vitamin Solution and 1~10ml / L ModifiedWolfe's Minerals; the solvent in the culture medium is water; Formulation 3: 0.5 g / L peptone, 0.5 g / L yeast extract, 1 g / L potassium dihydrogen phosphate, 1 g / L sodium dihydrogen phosphate, 0.2 g / L sodium chloride, 1 g / L ammonium dihydrogen phosphate, 0.2 g / L calcium chloride, 1 g / L sodium bicarbonate, 5 g / L sodium acetate, 2 g / L sodium lactate, 1 g / L sodium citrate, 0.2 g / L cysteine, 1 g / L magnesium sulfate, 0.5 g / L low crystallinity iron oxide powder, 15-20 g / L agar powder, 10 ml / L Wolfe's Vitamin Solution and 10 ml / L Modified Wolfe's Minerals; the solvent in the culture medium is water; Formulation 4: 0.05 g / L peptone, 0.05 g / L yeast extract, 1 g / L potassium dihydrogen phosphate, 1 g / L sodium dihydrogen phosphate, 1 g / L magnesium chloride, 0.2 g / L sodium chloride, 1 g / L ammonium dihydrogen phosphate, 0.2 g / L calcium chloride, 1 g / L sodium bicarbonate, 5 g / L sodium acetate, 2 g / L sodium lactate, 1 g / L sodium citrate, 0.2 g / L L-cysteine, 2 g / L sulfur powder, 2 g / L low crystallinity iron oxide powder, 10 ml / L Wolfe's Vitamin Solution and 10 ml / L Modified Wolfe's Minerals; the solvent in the culture medium is water; Formulation 5: 0.5 g / L peptone, 0.5 g / L yeast extract, 1 g / L potassium dihydrogen phosphate, 1 g / L sodium dihydrogen phosphate, 0.2 g / L sodium chloride, 1 g / L ammonium dihydrogen phosphate, 0.2 g / L calcium chloride, 1 g / L sodium bicarbonate, 5 g / L sodium acetate, 2 g / L sodium lactate, 1 g / L sodium citrate, 0.2 g / L L-cysteine, 1 g / L magnesium sulfate, 2 g / L low crystallinity iron oxide powder, 0.5 g / L ferrous chloride, 10 ml / L Wolfe's Vitamin Solution and 10 ml / L Modified Wolfe's Minerals; the solvent in the culture medium is water.

6. A method for preparing a culture medium, comprising the following steps: Deoxygenating the mixture A, sterilizing it under high pressure, cooling it, and then mixing it with the mixture B to obtain the culture medium; The mixture A comprises 0.05-2.5 g / L peptone, 0.05-2.5 g / L yeast extract, 0.02-1 g / L sodium chloride, 0.3-15 g / L phosphate, 0.5-25 g / L sodium acetate, 0.02-1 g / L calcium chloride and 0.01-20 g / L electron acceptor; The mixture B comprises 0-5 g / L sodium citrate, 0.2-10 g / L sodium lactate, 0.1-5 g / L sodium bicarbonate and 0.02-1 g / L L-cysteine; the solvent in the culture medium is water; wherein g / L represents the ratio of the mass of each component to the total volume of the culture medium; When the electron acceptor is manganese dioxide, or iron oxide and / or a sulfur-containing reagent; the total amount of phosphate, sodium chloride, sodium citrate, sodium lactate, calcium chloride and sodium bicarbonate in the mixture A and the mixture B does not exceed 15 g / L; When the electron acceptor is dichromate, the content of the electron acceptor is 0.01-5 g / L; the total amount of phosphate, sodium chloride, sodium citrate, sodium lactate, calcium chloride and sodium bicarbonate in the mixture A and mixture B, and the dichromate does not exceed 15 g / L.

7. The preparation method according to claim 6, characterized in that: It meets one or more of the following conditions: (1) The mixture B also includes 1-50 ml / L Wolfe's Vitamin Solution; (2) The mixture B also includes 1-50 ml / L Modified Wolfe's Minerals; (3) The mixed solution A also includes 1.5-30 g / L agar powder; (4) The pH of the culture medium is 6.5-8; (5) The content of peptone is 0.05-1.3 g / L; (6) The yeast extract has a content of 0.05 to 1.3 g / L; (7) The phosphate is 0.1-5 g / L potassium dihydrogen phosphate, 0.1-5 g / L sodium dihydrogen phosphate and 0.1-5 g / L ammonium dihydrogen phosphate; (8) The content of L-cysteine ​​is 0.2 g / L; (9) The water is distilled water or deionized water; (10) When the electron acceptor is dichromate, the dichromate is potassium dichromate; (11) When the electron acceptor is manganese dioxide, the content of manganese dioxide is 2.5 g / L; (12) The iron oxide is low crystallinity iron oxide; (13) The sulfur-containing reagent is sulfate or sulfur powder, preferably magnesium sulfate, potassium sulfate or sulfur powder; (14) When the electron acceptor is iron oxide and / or a sulfur-containing reagent, the culture medium further comprises ferrous ions, and the ferrous ions are preferably ferrous chloride; for example, 0.05-2.5 g / L ferrous chloride; for example, 0.5 g / L ferrous chloride; (15) The culture medium is deoxygenated by passing a mixture of an inert gas and carbon dioxide; (16) In the mixed gas, the volume ratio of the inert gas to the carbon dioxide is (60-90): (40-10); (17) The temperature of the high pressure sterilization is 110~135℃; (18) The high pressure sterilization time is 10 to 30 minutes; (19) The cooling temperature is 10~50℃.

8. The preparation method according to claim 7, characterized in that: It meets one or more of the following conditions: (1) The culture medium further comprises 1-10 ml / L Wolfe's Vitamin Solution; (2) The culture medium further comprises 1-10 ml / L Modified Wolfe's Minerals; (3) The culture medium also includes 15-20 g / L agar powder; (4) The content of the peptone is 0.5 g / L, 0.55 g / L or 1.25 g / L; (5) The yeast extract content is 0.5 g / L, 0.55 g / L or 1.25 g / L; (6) The phosphate is 1 g / L potassium dihydrogen phosphate, 1 g / L sodium dihydrogen phosphate and 1 g / L ammonium dihydrogen phosphate; (7) When the electron acceptor is dichromate, the dichromate is 0.1-5 g / L potassium dichromate; (8) When the electron acceptor is dichromate, the pH of the culture medium is 7-8; (9) When the electron acceptor is manganese dioxide, the manganese dioxide is prepared by the following method: grinding manganese dioxide powder using a ball mill and sieving through a 600 mesh screen; (10) The low crystallinity iron oxide is prepared by the following method: grinding the iron oxide powder using a ball mill and sieving it through a 600 mesh screen; (11) When the electron acceptor is a combination of iron oxide and a sulfur-containing reagent, the content of the electron acceptor is 0.15-7.5 g / L, 0.3-15 g / L or 0.4-20 g / L; wherein the content of the iron oxide is 0.05-10 g / L, such as 0.5 g / L or 2 g / L; the content of the sulfur-containing reagent is 0.1-10 g / L; preferably 0.5-2 g / L; such as 1 g / L or 2 g / L; (12) When the electron acceptor is a combination of iron oxide and sulfur powder, the culture medium further comprises 0.1-5 g / L magnesium chloride, for example 1 g / L magnesium chloride; (13) The culture medium is deoxygenated by passing a mixture of an inert gas and carbon dioxide, wherein the inert gas is nitrogen; (14) In the mixed gas, the volume ratio of the inert gas to the carbon dioxide is 80:20; (15) The temperature of the high pressure sterilization is 121°C; (16) The high pressure sterilization time is 15 minutes; (17) The cooling temperature is 25°C or 45°C.

9. A culture medium prepared by the preparation method according to any one of claims 6 to 8.

10. Use of the culture medium according to any one of claims 1 to 5 or claim 9 in screening and purifying reducing bacteria having conductive pili; Preferably, the reducing bacteria of the conductive pili are Geobacter reductus, sulfate-reducing bacteria or metal-reducing bacteria.

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