A culture medium for efficiently screening and culturing olfactory actinomycetes in surface water

Abstract

The present invention provides a culture medium for efficiently screening and cultivating olfactory actinomycetes in surface water, and relates to the technical field of actinomycete cultivation. The culture medium is prepared by adding aspartic acid and methyl pyruvate to Gao's No. 1 culture medium to simplify the metabolic pathway in actinomycete cells, thereby improving the culture efficiency and growth rate of actinomycetes and efficiently screening and cultivating olfactory actinomycetes in surface water; the mass ratio of Gao's No. 1 culture medium to aspartic acid and methyl pyruvate is 20: (0.8-1.5): (0.2-0.6). The solid culture medium of the present invention can make dormant spores and cells with low metabolic levels in water bodies more directly and easily utilized, so that more actinomycetes can survive on the culture medium to improve the separation efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of actinomycete cultivation, and in particular to a culture medium for efficiently screening and cultivating olfactory actinomycetes in surface water. Background Art

[0002] Actinomycetes are a class of prokaryotic microorganisms widely found in soil and water, with extremely high diversity and potential biological activity. Actinomycetes are known for their spore reproduction and mycelial growth characteristics, and are divided into genera such as Actinomycetes, Nocardia, Micromonas, and Streptomyces. Early studies focused on actinomycetes in soil and marine environments, but as more and more scholars have discovered the existence of more actinomycetes in water environments such as rivers and lakes, research and attention have been raised on actinomycetes in water environments. In order to isolate and culture these actinomycetes, researchers continue to develop improved culture medium formulas.

[0003] Although the culture conditions of actinomycetes are relatively simple and do not require overly complex culture media and equipment, traditional culture methods usually take a long time to obtain sufficient actinomycete suspensions. Currently, widely used actinomycete culture media include Gao's medium No. 1, humic acid agar medium, International Streptomyces Project Medium 2, proline medium, etc., which play an important role in the separation and cultivation of actinomycetes. At present, the nutrients of the culture medium are mainly carbon sources, nitrogen sources, growth factors, trace elements and water, etc., and these nutrients are only determined according to the basic life metabolism of microorganisms. The tricarboxylic acid cycle is a metabolic pathway that is ubiquitous in aerobic organisms. Under aerobic conditions, pyruvate is oxidized and decarboxylated to generate acetyl CoA under the catalysis of the pyruvate dehydrogenase system, and the latter is then oxidized to CO2, H2O and produces energy through the tricarboxylic acid cycle. Various carbon sources enter the tricarboxylic acid cycle through the products of microbial metabolism. However, the metabolic levels and carbon source utilization capabilities of various microorganisms are different. For actinomycetes, which have a slower metabolism than common bacteria, these culture media can be used to selectively isolate actinomycetes, but their culture efficiency still needs to be improved. Generally, bacteria can grow colonies after 1 to 5 days of culture, while actinomycetes can grow colonies after 7 days of culture at 28°C. Especially in water environments, the screening and culture efficiency of actinomycetes will be slower.

[0004] The problem of odor in the water environment has been a research hotspot in the water supply field in recent years. In the aquatic ecosystem, the microbial community and its metabolic functions and pathways are complex and diverse. Many prokaryotic and eukaryotic microorganisms can metabolize and produce odorous compounds, such as more than 40 algae, actinomycetes and myxobacteria, such as Anabaena pseudospermum, filamentous cyanobacteria, Root algae and Oscillatoria. Actinomycetes are the main producers of odorous substances. There are many kinds of odorous compounds produced by their metabolism. So far, 67 kinds have been reported, with 2-methylisoborneol (2-MIB) and geosmin (trans-1,10-dimethyl-trans-9-decalol, GSM) being the main research. Even if these odorous compounds exist in trace amounts in the water environment, they will produce a relatively strong odor, thereby causing water environment safety and health incidents. Early studies have shown that physical, chemical and biological treatment of actinomycetes in water source reservoirs can effectively reduce the number of actinomycetes in water bodies and thus reduce the odor threshold concentration.

[0005] In the study of odor-producing actinomycetes, most scholars use actinomycete selection culture media to screen environmental samples, make preliminary judgments based on the cultured colony morphology and olfactory characteristics, and then use gene sequencing technology to identify odor genes to screen out odor-producing actinomycetes. The culture media used in the entire screening process are designed based on the metabolic characteristics and nutritional requirements of actinomycetes. These culture media can only specifically isolate actinomycetes from the environment, and then use this type of culture media to enrich and culture them in subsequent studies. However, the culture capabilities of various existing culture media are similar and relatively general, and it takes a long time of 10 days to reach a higher cell concentration for subsequent experimental research.

[0006] Therefore, it is necessary to provide a culture medium that can rapidly expand the culture of odor-producing actinomycetes. Summary of the invention

[0007] In order to solve the problem that it takes too long to cultivate olfactory actinomycetes in the culture medium in the prior art, the present invention provides a culture medium for efficiently screening and cultivating olfactory actinomycetes in surface water.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows.

[0009] The first aspect of the present invention provides a culture medium for efficiently screening and culturing olfactory actinomycetes in surface water. The culture medium is prepared by adding aspartic acid and methyl pyruvate to Gao's medium No. 1, using aspartic acid and methyl pyruvate to simplify the metabolic pathway in actinomycete cells, thereby improving the culture efficiency and growth rate of actinomycetes, and efficiently screening and culturing olfactory actinomycetes in surface water.

[0010] The mass ratio of the basic culture medium to aspartic acid and methyl pyruvate is 20:(0.8-1.5):(0.2-0.6).

[0011] The invention adds aspartic acid and methyl pyruvate as backup carbon sources to simplify the metabolic pathway in actinomycete cells, thereby improving the culture efficiency and growth rate of actinomycetes. Whether it is a liquid culture medium or a solid culture medium, the growth of actinomycetes is better than that of a common culture medium.

[0012] In another preferred embodiment, the culture medium for efficiently screening and culturing olfactory actinomycetes in surface water also includes B vitamins and nalidixic acid; the B vitamins are a mixture of niacin, inositol, biotin and p-aminobenzoic acid;

[0013] The mass ratio of niacin, inositol, biotin and p-aminobenzoic acid is 1:1:1:1.

[0014] In another preferred embodiment, in each liter of the culture medium for efficiently screening and culturing olfactory actinomycetes from surface water, the concentrations of nicotinic acid, inositol, biotin and p-aminobenzoic acid are all 0.5 mg / L, and the concentration of nalidixic acid is 25 mg / L.

[0015] In another preferred embodiment, the culture medium for efficiently screening and culturing olfactory actinomycetes in surface water is a solid culture medium or a liquid culture medium.

[0016] In another preferred embodiment, each liter of Gao's medium No. 1 comprises the following raw materials:

[0017] Soluble starch 20g / L, potassium nitrate 1.00g / L, potassium dihydrogen phosphate 0.5g / L, magnesium sulfate 0.5g / L, sodium chloride 0.5g / L, ferrous sulfate 0.01g / L.

[0018] The second aspect of the present invention provides a method for preparing a culture medium for efficiently screening and culturing olfactory actinomycetes in surface water. The culture medium for efficiently screening and culturing olfactory actinomycetes in surface water is provided by the first aspect of the present invention, and comprises the following steps:

[0019] After adding aspartic acid, methyl pyruvate, B vitamins and nalidixic acid into Gao's No. 1 culture medium and mixing, the pH value is adjusted to 7.2-8.0, and sterilization is performed to obtain the culture medium for efficiently screening and cultivating olfactory actinomycetes in surface water.

[0020] In another preferred embodiment, when the culture medium for efficiently screening and culturing olfactory actinomycetes in surface water is a solid culture medium, the specific preparation method is as follows:

[0021] Dilute methyl pyruvate with pure water to 0.4 g / L, sterilize, and obtain diluted methyl pyruvate;

[0022] After aspartic acid is mixed with Gao's medium No. 1, vitamin B is added to adjust the pH value to 7.2-8.0. After the medium is sealed and sterilized, it is poured onto a flat plate culture dish, and after it is cooled, 10-200 μL of diluted methyl pyruvate is added to the surface to obtain.

[0023] When the culture medium for efficiently screening and culturing olfactory actinomycetes in surface water is a solid culture medium, after adding aspartic acid and methyl pyruvate, the CFU number of actinomycetes in the screened water source reservoir is increased by 1.3 to 2.0 times compared with the ordinary culture medium.

[0024] In another preferred embodiment, when the culture medium for efficiently screening and culturing olfactory actinomycetes in surface water is a liquid culture medium, the preparation method of the liquid culture medium is as follows:

[0025] Aspartic acid, methyl pyruvate, B vitamins and nalidixic acid are added to Gao's medium No. 1, the mixture is adjusted to a pH value of 7.2 to 8.0, and sterilized to obtain the product.

[0026] When the culture medium for efficiently screening and culturing olfactory actinomycetes in surface water is a liquid culture medium, the cell concentration of enriched actinomycetes in the same culture time is one order of magnitude higher than that of ordinary culture medium.

[0027] In another preferred embodiment, the sterilization pressure is 103 kPa to 205 kPa, and the time is 15 to 35 minutes.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The culture medium for efficiently screening and cultivating olfactory actinomycetes from surface water provided by the present invention adds aspartic acid and methyl pyruvate as backup carbon sources to Gao's No. 1 culture medium, thereby simplifying the metabolic pathway in the actinomycete cells, significantly improving the cultivation efficiency and growth rate of the actinomycetes, and effectively solving the problem of slow cultivation speed of actinomycetes with current culture medium.

[0030] (2) The solid culture medium provided by the present invention is used to screen and culture aquatic actinomycetes. After 7 days of incubation, the number of colonies cultured on the solid culture medium provided by the present invention is about 30% to 45% higher than that of Gao's No. 1. This is mainly due to the addition of aspartic acid and methyl pyruvate, which makes it more directly and easily available for dormant spores and cells with low metabolic levels in the water, thereby enabling more actinomycetes to survive on the culture medium, thereby improving the separation efficiency.

[0031] (3) The liquid culture medium configured with the actinomycete culture medium formula provided by the present invention, after inoculation of bacterial liquid and 7 days of enrichment culture, the obtained bacterial liquid concentration is significantly higher than Gao's No. 1 and other common culture media. Even for Streptomyces, the cell concentration after 3 days of culture can be one order of magnitude higher than the number of cells in other culture media. In addition to the soluble starch preferred by actinomycetes, aspartic acid, as a precursor substance for many important biological molecules, greatly simplifies the metabolic process of actinomycetes in the liquid culture medium. In addition, methyl pyruvate is directly decomposed into pyruvate and participates in the tricarboxylic acid cycle, which also greatly improves the growth rate of actinomycetes in the early stage of culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a comparison chart of the CFU of actinomycetes in the culture medium for efficient screening and cultivation of olfactory actinomycetes in surface water in Example 1 of the present invention and the typical actinomycete culture medium Gao's No. 1 screening and cultivation water source reservoir.

[0033] Figure 2 This is a comparison chart of the CFU of actinomycetes in water source reservoirs screened using seven typical actinomycete culture media.

[0034] Figure 3 This is a comparison chart of the number of Streptomyces cultured in the culture medium for efficient screening and cultivation of olfactory actinomycetes in surface water in Example 2 of the present invention and three typical actinomycete culture media.

[0035] Figure 4 This is a comparison chart of the number of Amycolatopsis cultured using a culture medium for efficient screening and cultivation of olfactory actinomycetes in surface water in Example 2 of the present invention and three typical actinomycete culture media. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0037] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work shall fall within the scope of protection of the present invention.

[0038] Example 1

[0039] A culture medium for efficiently screening and cultivating olfactory actinomycetes in surface water, wherein the concentration of each raw material in each liter of the culture medium is:

[0040] Soluble starch 20g / L, potassium nitrate 1g / L, aspartic acid 1.2g / L, methyl pyruvate 0.4g / L, ferrous sulfate 0.01g / L, magnesium sulfate 0.5g / L, dipotassium hydrogen phosphate 0.5g / L, sodium chloride 0.5g / L, niacin 0.5mg / L, inositol 0.5mg / L, biotin 0.5mg / L and p-aminobenzoic acid 0.5mg / L, nalidixic acid 25mg / L;

[0041] The method for preparing the culture medium for efficiently screening and culturing olfactory actinomycetes in surface water comprises the following steps:

[0042] Methyl pyruvate was diluted to 0.4 g / L with pure water. The other components except methyl pyruvate were weighed according to the above concentrations and added to pure water to prepare the culture medium matrix. The two were respectively mixed at 1×10 5 Sterilize under Pa conditions for 30 min.

[0043] The culture medium matrix was distributed into a flat plate and allowed to stand overnight, and 100 μL of methyl pyruvate dilution was evenly spread on the surface of the culture medium to obtain a solid culture medium for efficiently screening and culturing olfactory actinomycetes in surface water.

[0044] Example 2

[0045] A culture medium for efficiently screening and cultivating olfactory actinomycetes in surface water, wherein the concentration of each raw material in each liter of the culture medium is:

[0046] Soluble starch 20g / L, potassium nitrate 1g / L, aspartic acid 1.2g / L, methyl pyruvate 0.4g / L, ferrous sulfate 0.01g / L, magnesium sulfate 0.5g / L, dipotassium hydrogen phosphate 0.5g / L, sodium chloride 0.5g / L, nicotinic acid 0.5mg / L, inositol 0.5mg / L, biotin 0.5mg / L and p-aminobenzoic acid 0.5mg / L, nalidixic acid 25mg / L, agar powder 20g / L;

[0047] The method for preparing the culture medium for efficiently screening and culturing olfactory actinomycetes in surface water comprises the following steps:

[0048] Weigh each raw material in the culture medium according to the above concentrations and add 1×10 5 After sterilization for 30 minutes under Pa conditions and cooling to room temperature, a liquid culture medium for efficiently screening and culturing olfactory actinomycetes from surface water was obtained.

[0049] In order to further illustrate that the culture medium for efficiently screening and culturing odor-producing actinomycetes in surface water in the present invention, whether it is a liquid culture medium or a solid culture medium, can quickly expand the cultivation of odor-producing actinomycetes, we conducted the following experiments.

[0050] The screening process of the following Streptomyces and Amycolatopsis is as follows: the water sample of the water source reservoir is enriched on a 0.22 micron filter membrane, the filter membrane is inverted on an actinomycete isolation medium, potassium dichromate is added to the medium, after incubation at 28°C for 7 days, the colonies on the medium are selected, and the colonies are purified by the plate streaking method, and pure bacteria are obtained after purification 5-7 times. After the strain identification, one strain of Streptomyces and one strain of Amycolatopsis were selected.

[0051] 1. Cultivation of actinomycetes by the medium in Example 1 and Gao's medium No. 1

[0052] 1) Water sampling: Use a sampler to collect water near the drinking water tower of Lijiahe Reservoir. Rinse the sampler three times with water from the reservoir before sampling. Lower the sampler to the middle layer of the water body, i.e. 30m-45m. After the sampler is filled, quickly retract the sampler. Store the collected water sample in a sterilized polyethylene water bottle and keep the temperature at about 4°C. Bring it back to the laboratory within 24 hours and conduct a colony counting experiment.

[0053] 2) Determination of actinomycete abundance in water source reservoirs:

[0054] The culture medium prepared in Example 1 was selected, referred to as WS; and the WS culture medium and Gao's medium No. 1 were used as control groups to measure the cell number:

[0055] Gao's medium No. 1: soluble starch 20g / L, potassium nitrate 1.00g / L, potassium dihydrogen phosphate 0.5g / L, magnesium sulfate 0.5g / L, sodium chloride 0.5g / L, ferrous sulfate 0.01g / L; weigh the raw materials in the medium according to the above concentrations and add them to 1×10 5 Sterilize under Pa conditions for 30 min.

[0056] K2Cr2O7 solution was added to each culture medium to make the concentration 50 mg / L. Before cooling, it was distributed into round culture dishes and cooled to room temperature for use.

[0057] 3) Determination of actinomycete abundance

[0058] In the present invention, membrane filtration and pure culture counting are used to perform enrichment culture of culturable actinomycetes and determine the abundance of actinomycetes. Use a 0.22μm polycarbonate membrane to filter 100mL of water sample, and the water sample should be fully shaken and mixed before filtration. Carefully remove the filter membrane and invert it on the solid culture medium, and wrap it with a sealing film to avoid contamination by other bacteria. Incubate the solid culture medium at 30±2°C for 7 days to ensure that all actinomycetes can grow well. The number of actinomycetes is determined by counting colony forming units (CFU). Three parallel samples (n=3) are set for each sample, and the results are as follows Figure 1 As shown. Figure 1As shown, two water samples, group A and group B, were collected respectively, and culture counting experiments were carried out respectively. The results of culture using the medium prepared by the formula in Example 1 of the present invention, namely WS medium and Gao's No. 1, were compared. Obviously, the number of culturable colonies in the two groups of water samples on WS medium was significantly higher than that of Gao's No. 1. From the results of the culture of the water samples in group A, the average number of culturable colonies in WS medium reached about 1400 CFU / L, while the number of culturable colonies in ordinary Gao's No. 1 was only about 1100 CFU / L (p=0.04); such differences were more obvious in the water samples in group B (p=0.005), the average number of culturable colonies in WS medium was about 1000 CFU / L, while the number of culturable colonies in ordinary Gao's No. 1 was only about 700 CFU / L. The culture results of the two groups showed that the number of colonies screened by WS medium was about 30% to 45% higher than that of Gao's No. 1.

[0059] like Figure 2 As shown in the figure, the excellent performance of Gao's No. 1 in the screening culture of actinomycetes in water bodies was verified in the previous research process. That is, when 7 typical actinomycete culture media were used to screen the same water sample, the number of colonies cultured by Gao's No. 1 was significantly higher than that of other culture media.

[0060] The 7 typical actinomycete culture media are as follows:

[0061] Gao's medium No. 1: soluble starch 20g / L, potassium nitrate 1.00g / L, potassium dihydrogen phosphate 0.5g / L, magnesium sulfate 0.5g / L, sodium chloride 0.5g / L, ferrous sulfate 0.01g / L;

[0062] Proline medium: L-proline 10.0 g / L, dipotassium hydrogen phosphate 0.08 g / L, magnesium sulfate 0.20 g / L, potassium dihydrogen phosphate 0.02 g / L;

[0063] Humic acid culture medium: humic acid 1g / L, potassium chloride 1.76g / L, ferrous sulfate 0.01g / L, magnesium sulfate 0.05g / L, disodium hydrogen phosphate 0.5g / L, calcium chloride 1g / L;

[0064] LB medium: tryptone 0.1 g / L, 0.5% yeast extract 0.025 g / L, sodium chloride 0.1 g / L;

[0065] Aspartic acid-glycerol medium: aspartic acid 1.00 g / L, potassium nitrate 5.00 g / L, dipotassium hydrogen phosphate 1.00 g / L, yeast extract 5.00 g / L, glycerol 10 g / L;

[0066] Raffinose-histidine medium: raffinose 10g / L, L-histidine 0.1g / L, ferrous sulfate 0.1g / L, magnesium sulfate 0.5g / L, potassium hydrogen phosphate 1g / L;

[0067] Mannitol culture medium: mannitol 0.2g / L, casein 1g / L, dipotassium hydrogen phosphate 0.g / L7, potassium dihydrogen phosphate 0.5g / L.

[0068] 2. Cultivation of actinomycetes using three culture media

[0069] Preparation of Streptomyces suspension:

[0070] Streptomyces was added to Gao's No. 1 liquid culture medium for enrichment culture for 7 days, and then the culture medium was centrifuged at 4°C, 8000 rpm for 10 minutes, the supernatant was discarded, PBS solution was added to the centrifuge tube, mixed thoroughly, and then centrifuged under the same conditions for 10 minutes, the supernatant was discarded, and this process was repeated three times. After the supernatant was discarded for the last time, PBS was added and mixed thoroughly for later use.

[0071] Determination of the number of Streptomyces:

[0072] The culture medium prepared in Example 2 was selected, and the culture medium in Example 2 and three typical culture media commonly used at present were used as control groups to measure the number of cells:

[0073] Gao's medium No. 1: soluble starch 20g / L, potassium nitrate 1.00g / L, potassium dihydrogen phosphate 0.5g / L, magnesium sulfate 0.5g / L, sodium chloride 0.5g / L, ferrous sulfate 0.01g / L;

[0074] Proline medium: L-proline 10.0 g / L, dipotassium hydrogen phosphate 0.08 g / L, magnesium sulfate 0.20 g / L, potassium dihydrogen phosphate 0.02 g / L;

[0075] Aspartic acid-glycerol medium: aspartic acid 1.00 g / L, potassium nitrate 5.00 g / L, dipotassium hydrogen phosphate 1.00 g / L, yeast extract 5.00 g / L, glycerol 10 g / L;

[0076] The cell number test method is as follows: the sterilized liquid culture medium is dispensed into high-pressure sterilized triangular flasks, and then the prepared Streptomyces suspension is diluted to a concentration of 10 using phosphate buffer solution. 5 to 10 6Within the range, pipette 1mL of the suspension into a triangular flask, mix thoroughly and seal the bottle mouth with a sealing film. Move each triangular flask to a shaking incubator for constant temperature culture, and measure the cell concentration after the third and seventh days of culture. Pipette the bacterial suspension in the triangular flask into a sterilized 10mL centrifuge tube, add phosphate buffer solution to the 10mL mark, shake and mix thoroughly, repeat this method to dilute the original bacterial suspension 1000 times and use it to determine the cell concentration. Pipette 245μL of the diluted cell suspension into a 1.5mL conical bottom centrifuge tube, add 5μLPI / GS dye, shake and mix thoroughly, place in a 35℃ metal bath for 20 minutes, and use a flow cytometer to measure the cell concentration and calculate the cell concentration of the original bacterial solution. Set up three triangular flasks for each culture medium for parallel samples and take the average value.

[0077] Cell concentration (cells / mL): N / 30×1000×dilution factor (where N is the cell concentration measured by flow cytometry);

[0078] The results are as follows Figure 3 As shown in the figure, in terms of cell concentration, there is a significant difference between WS medium and the other three mediums. After three days of culture, the concentration of Streptomyces in Gao's medium No. 1 has not reached 1.0×10 8 The cell concentration of WS culture medium has reached 5.0×10 8 The cell concentration was even higher than that of Gao's No. 1 and proline medium after seven days of culture. By the seventh day of culture, the number of cells in the WS new medium had reached 4.2×10 9 about.

[0079] 3) Cultivation of Pseudomonas amylovora

[0080] Preparation of pseudoamycarboxylic acid suspension:

[0081] Amorphic acid was added to Gao's No. 1 liquid culture medium for enrichment culture for 7 days, and then the culture medium was centrifuged at 4°C, 8000 rpm for 10 minutes, the supernatant was discarded, PBS solution was added to the centrifuge tube, mixed thoroughly, and then centrifuged for 10 minutes under the same conditions, the supernatant was discarded, and this process was repeated three times. After the supernatant was discarded for the last time, PBS was added and mixed thoroughly for use.

[0082] Determination of the number of Amycolatopsis spp.

[0083] The culture medium prepared in Example 2 was selected, and the culture medium in Example 2 and three typical culture media commonly used at present were used as control groups to measure the number of cells:

[0084] Gao's medium No. 1: soluble starch 20g / L, potassium nitrate 1.00g / L, potassium dihydrogen phosphate 0.5g / L, magnesium sulfate 0.5g / L, sodium chloride 0.5g / L, ferrous sulfate 0.01g / L;

[0085] Proline medium: L-proline 10.0 g / L, dipotassium hydrogen phosphate 0.08 g / L, magnesium sulfate 0.20 g / L, potassium dihydrogen phosphate 0.02 g / L;

[0086] Aspartic acid-glycerol medium: aspartic acid 1.00 g / L, potassium nitrate 5.00 g / L, dipotassium hydrogen phosphate 1.00 g / L, yeast extract 5.00 g / L, glycerol 10 g / L;

[0087] The cell number test method is as follows: the sterilized liquid culture medium is dispensed into high-pressure sterilized triangular flasks, and then the freshly prepared pseudo-amylotic acid suspension is diluted to a concentration of 10 using phosphate buffer solution. 5 to 10 6 Within the range, pipette 1mL of the suspension into a triangular flask, mix thoroughly and seal the bottle mouth with a sealing film. Move each triangular flask to a shaking incubator for constant temperature culture, and measure the cell concentration after the third and seventh days of culture. Pipette the bacterial suspension in the triangular flask into a sterilized 10mL centrifuge tube, add phosphate buffer solution to the 10mL mark, shake and mix thoroughly, repeat this method to dilute the original bacterial suspension 100 times and use it to determine the cell concentration. Pipette 245μL of the diluted cell suspension into a 1.5mL conical bottom centrifuge tube, add 5μLPI / GS dye, shake and mix thoroughly, place in a 35℃ metal bath for 20 minutes, and use a flow cytometer to measure the cell concentration and calculate the cell concentration of the original bacterial solution. Set up three triangular flasks for each culture medium for parallel samples and take the average value.

[0088] Cell concentration (cells / mL): N / 30×1000×dilution factor (where N is the cell concentration measured by flow cytometry);

[0089] The results are as follows Figure 4 As shown in the figure, compared with Streptomyces, Pseudomonas amycoticus does not have a very typical branched hyphae structure similar to Streptomyces, but in terms of cell concentration, the culture medium of Example 2 is more efficient than the other three culture media. After three days of culture, the cell concentrations of the four culture media were similar, and Gao's No. 1 culture medium had a higher cell concentration. On the seventh day of culture, the number of cells in the culture medium of Example 2 had reached 3.0×10 9 The cell concentration of its culture is also one to two times higher than that of the other three culture media.

[0090] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A culture medium for screening and culturing olfactory actinomycetes in surface water, characterized in that: The culture medium is prepared by adding aspartic acid and methyl pyruvate to Gao's medium No. 1 to simplify the metabolic pathway in the actinomycete cells, thereby improving the culture efficiency and growth rate of the actinomycetes, so as to screen and culture the olfactory actinomycetes in the surface water. The mass ratio of Gao's medium No. 1 to aspartic acid and methyl pyruvate is 20:(0.8-1.5):(0.2-0.6) ； The culture medium for culturing olfactory actinomycetes in surface water also includes B vitamins and nalidixic acid; the B vitamins are a mixture of nicotinic acid, inositol, biotin and p-aminobenzoic acid; the mass ratio of nicotinic acid, inositol, biotin and p-aminobenzoic acid is 1:1:1:1; In each liter of the culture medium for culturing olfactory actinomycetes from surface water, the concentrations of nicotinic acid, inositol, biotin and p-aminobenzoic acid are all 0.5 mg / L, and the concentration of nalidixic acid is 25 mg / L.

2. The culture medium for culturing olfactory actinomycetes in surface water according to claim 1, characterized in that: The culture medium for screening and culturing olfactory actinomycetes in surface water is a solid culture medium or a liquid culture medium.

3. The culture medium for culturing olfactory actinomycetes in surface water according to claim 1, characterized in that: Each liter of Gao's medium No. 1 includes the following raw materials: Soluble starch 20g / L, potassium nitrate 1.00g / L, potassium dihydrogen phosphate 0.5g / L, magnesium sulfate 0.5g / L, sodium chloride 0.5g / L, ferrous sulfate 0.01g / L.

4. A method for preparing a culture medium for culturing olfactory actinomycetes in surface water according to any one of claims 1 to 3, characterized in that: The following steps are involved: Aspartic acid, methyl pyruvate, B vitamins and nalidixic acid are added to Gao's medium No. 1 and mixed, and the pH value is adjusted to 7.2-8.0, and sterilized to obtain the culture medium for screening and culturing olfactory actinomycetes in surface water.

5. The method for preparing a culture medium for screening and cultivating olfactory actinomycetes in surface water according to claim 4, characterized in that: When the culture medium for culturing olfactory actinomycetes in surface water is a solid culture medium, the specific preparation method is as follows: Dilute methyl pyruvate with pure water to 0.4 g / L, sterilize, and obtain diluted methyl pyruvate; Aspartic acid, Gao's medium No. 1, B vitamins and nalidixic acid are mixed, the pH value is adjusted to 7.2-8.0, sterilized, and 10-200 μL of diluted methyl pyruvate is added to obtain.

6. The method for preparing a culture medium for screening and cultivating olfactory actinomycetes in surface water according to claim 4, characterized in that: When the culture medium for culturing olfactory actinomycetes in surface water is a liquid culture medium, the specific preparation method is as follows: Aspartic acid, methyl pyruvate, B vitamins and nalidixic acid are added to Gao's medium No. 1, the mixture is adjusted to a pH value of 7.2 to 8.0, and sterilized to obtain the product.

7. The method for preparing a culture medium for culturing olfactory actinomycetes in surface water according to claim 6, characterized in that: The sterilization pressure is 103 kPa to 205 kPa, and the time is 15 to 35 minutes.

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