CN111235071B - Rhodopseudomonas palustris culture medium and application thereof - Google Patents
Rhodopseudomonas palustris culture medium and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of microbial culture, and particularly relates to a rhodopseudomonas palustris culture medium and application thereof. The composition and formula of the health food comprises citric acid, beef extract, yeast extract, peptone, sodium acetate, magnesium sulfate, ferrous sulfate, and VB 6 Calcium chloride, potassium dihydrogen phosphate and a composite inorganic salt solution, wherein the solvent is water. By optimizing the formula, the quality of the rhodopseudomonas palustris microbial inoculum is greatly improved, so that the rhodopseudomonas palustris microbial inoculum has the advantages of high effective viable count, positive color, no layering, low mixed bacteria rate, stable quality and good preservation effect, and the rhodopseudomonas palustris microbial inoculum cultured by using the culture medium obtains good effect when being applied to aquaculture.
Description
Technical Field
The invention belongs to the technical field of microbial culture, and particularly relates to a rhodopseudomonas palustris culture medium and application thereof.
Background
Rhodopseudomonas palustris belongs to the family of Rhodospirillaceae and the genus Rhodopseudomonas and is one of the most widely studied and applied photosynthetic bacteria at present. Photosynthetic bacteria are prokaryotes which appear earliest on the earth, commonly exist in the nature and have an original light energy synthesis system, and are microorganisms which take light as energy and can use organic matters, sulfides, ammonia and the like in the nature as hydrogen donor and carbon source to carry out photosynthesis under the anaerobic illumination or aerobic dark condition. Photosynthetic bacteria are widely distributed in soil, paddy fields, swamps, lakes, rivers, seas and the like in the nature, and are mainly distributed in anoxic areas where light can transmit in aquatic environments. The rhodopseudomonas palustris is one of the feed-grade microorganisms allowed to be used by the Ministry of agriculture in China, and the preparation of the rhodopseudomonas palustris is widely applied to a plurality of fields of aquaculture, feed additives, sewage treatment and the like, and has great economic, ecological and social benefits.
In aquaculture, rhodopseudomonas palustris is put into a fish and shrimp culture pond, and particularly under the anoxic condition of bottom mud in a deep layer of the pond, organic waste can be decomposed, the concentration of hydrogen sulfide and ammonia in water can be reduced, and the cleanness of water quality is kept; in addition, the rhodopseudomonas palustris contains active substances and trace elements required by growth and development of fish and shrimp bodies, including bioactive substances such as protein, amino acid, B vitamins, coenzyme Q and the like, which are activators or coenzymes of enzymes, hormones and bioactive substances in the fish and shrimp bodies, so that the activity and the content of the enzymes and the bioactive substances can be changed, the growth and development of the fish and the shrimp are promoted, and the using amount of feed is reduced; moreover, the rhodopseudomonas palustris has the functions of resisting diseases and improving the immunity of organisms, and can effectively prevent aquatic organisms from generating diseases.
Patent CN104388364A discloses a fermentation medium of seawater photosynthetic bacteria, which comprises ammonium chloride, yeast extract powder, peptone, anhydrous sodium acetate, iron phosphate and seawater, and the examples show that OD is OD after 6 days of culture 660nm It was 1.635 (the fermentation yield to photosynthetic bacteria was expressed as the absorbance at 660nm, OD 660nm . ) (ii) a Patent CN105199993 discloses a photosynthetic bacteria culture medium and a preparation method thereof, the components are sodium acetate trihydrate, ammonium sulfate, sodium bicarbonate, potassium dihydrogen phosphate, magnesium sulfate and water, the photosynthetic bacteria OD is cultured by adopting the culture medium of the invention 660nm Is 2.2; patent CN107746821A discloses a photosynthetic bacteria culture medium, which comprises carbon-containing organic substance, ammonium chloride, yeast extract, peptone, magnesium chloride, calcium chloride, sodium dihydrogen acid, disodium hydrogen phosphate, manganese chloride, zinc sulfate and distilled water, wherein the OD after culture is shown in the examples 660nm Was 2.8.
Due to limited literature data on the intensive research on the high-density fermentation formula of the photosynthetic bacteria, the problems of low effective viable count, more mixed bacteria, poor product stability, short shelf life and the like commonly exist in the existing products on the market, and the application of the photosynthetic bacteria in aquaculture is limited.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a rhodopseudomonas palustris culture medium and application thereof, and the quality of a rhodopseudomonas palustris microbial inoculum is greatly improved through formula optimization, so that the rhodopseudomonas palustris microbial inoculum has the advantages of high effective viable count, positive color, no layering, low mixed bacteria rate, stable quality and good preservation effect, and the rhodopseudomonas palustris microbial inoculum cultured by using the culture medium obtains good effect when being applied to aquaculture.
Firstly, the rhodopseudomonas palustris culture medium comprises the following components in percentage by weight: 0.75-1.25g/L of citric acid, 1.50-3.00g/L of beef extract, 0.42-0.62g/L of yeast extract, 1.50-2.00g/L of peptone, 0.40-0.60g/L of sodium acetate, 0.25-0.50g/L of magnesium sulfate, and sulfite5.00-8.00mg/L of iron and VB 6 0.1-0.3mg/L, 0.10-0.30g/L of calcium chloride, 0.10-0.25g/L of monopotassium phosphate, 0.5-1.0mL/L of composite inorganic salt solution, water as solvent, and adjusting the pH value to 6.8-7.0.
The composite inorganic salt solution comprises: 0.5g/L of EDTA, 0.01g/L of zinc sulfate, 0.03g/L of boric acid, 0.003g/L of manganese chloride, 0.02g/L of calcium chloride, 0.001g/L of copper chloride, 0.003g/L of sodium molybdate and water as a solvent.
The citric acid is citric acid containing one crystal water, the sodium acetate is sodium acetate containing three crystal water, the magnesium sulfate is anhydrous magnesium sulfate, the ferrous sulfate is ferrous sulfate containing seven crystal water, the calcium chloride is anhydrous calcium chloride, and the potassium dihydrogen phosphate is anhydrous potassium dihydrogen sulfate.
Preferably, the rhodopseudomonas palustris culture medium adopts sodium hydroxide or potassium hydroxide with the mass concentration of 20% to adjust the pH.
Preferably, the potassium dihydrogen phosphate and the inorganic salt solution of the compound in the medium components need to be sterilized separately.
The inventor further utilizes the culture medium of the invention to research and obtain a culture method of rhodopseudomonas palustris fungicide, and the technical scheme is as follows: inoculating rhodopseudomonas palustris strain in culture medium for culturing rhodopseudomonas palustris for anaerobic culture, wherein the inoculum size is 10-50%, the illumination intensity is 3000-5000Lux, the temperature is 30-33 ℃, the culture time is 2-7 days, and the color is gradually changed from light red to red brown, namely the culture maturity. The macro pseudomonas aeruginosa strains selected by the culture method are all conventional commercial strains.
The more specific preparation steps of the microbial inoculum comprise (1) strain activation, (2) strain culture, (3) enlarged culture:
(1) Activating strains: inoculating the strain in a light-transmitting test tube culture medium, activating, culturing at constant temperature of 30-50% and illumination intensity of 3000-5000Lux for 3-5 days under sealed condition at 30-33 deg.C, wherein the color is changed from light red, red to red brown gradually to obtain mature culture;
preferably, the test tube should be rotated by shaking at least 1 time every day during the culture period, so that the rhodopseudomonas palustris can fully receive the light to be beneficial to growth.
Preferably, the total liquid content of the test tube is 90%.
(2) Seed culture: transferring the activated strain obtained in the step (1) to a light-transmitting culture container for culture, wherein the inoculation amount is 30-50%, the illumination intensity is 3000-5000Lux, the constant temperature of 30-33 ℃ is sealed and cultured for 2-4 days, and the color is changed from light red, red to reddish brown gradually, namely the culture maturity is obtained, and a seed solution is obtained;
(3) And (3) amplification culture: inoculating the seed liquid into a larger culture container, wherein the inoculation amount is 10-30%, and the culture conditions are as follows: the illumination intensity is 3000-5000Lux, the temperature is controlled at 30-33 ℃, and the culture is sealed for 5-7 days.
Preferably, all culture containers are subjected to chemical sterilization, specifically, 0.5 per mill of sodium hypochlorite aqueous solution is soaked for 12-24 hours or rinsed for 2 times by using 75% alcohol; sterilizing potassium dihydrogen phosphate, compound inorganic salt solution, and other rest components at 115 deg.C for 10 min.
The rhodopseudomonas palustris produced by the method and the rhodopseudomonas palustris culture medium special for aquaculture has high thallus density, and OD is obtained after the obtained bacterial liquid is diluted by 20 times by pure water 660nm Is 0.460, and has effective viable count of 8.96 × 10 9 One per mL. The method is applied to water plant culture, can quickly reduce the content of ammonia nitrogen and nitrite in the water body, effectively increases the dissolved oxygen in the water body, and greatly improves the yield of aquaculture.
Compared with the prior art, the invention has the following advantages:
ferrous sulfate and VB in the formula of the culture medium 6 The composite addition plays an important role in strain growth, photosynthetic bacteria can carry out circulating light and phosphorylation under the drive of light energy under the anaerobic condition through electron transfer, electrons are expelled from bacteriochlorin molecules and are received by the bacteriochlorin again through the circulating transfer of coenzyme Q, cytochrome bc1, iron-sulfur protein and cytochrome bc1, proton electromotive force is established and 1 ATP is generated in the process, and under the condition of supplying the ATP, the external electron donor can generate reducing force against electron flow, the light and phosphorylation are promoted to be connected with Calvin circulation, and carbon dioxide is fixed, so that the growth and the propagation of bacteria are promoted. In the present invention, ferrous sulfate and VB 6 Can circulate in photosynthetic bacteriaIn the process of ring photosynthetic phosphorylation, an effective exogenous electron donor generates reducing force against electron flow, light phosphorylation is promoted to be linked with Calvin circulation, and carbon dioxide is fixed, so that a large amount of thalli grow and reproduce, and secondly, the dosage of ferrous sulfate is moderate, and excessive ferrous ions can form ferrous sulfide, iron oxide and other products to make the microbial inoculum darker in color; the microbial inoculum produced by the formula has the advantages of normal color, no layering, short fermentation period, no increase of pH, thorough utilization of thalli on a culture medium, and effective inhibition of breeding of algae and other bacteria during fermentation. The rhodopseudomonas palustris microbial inoculum produced by the formula is applied to water plant culture, so that the content of ammonia nitrogen and nitrite in water can be quickly reduced, the dissolved oxygen in the water is effectively increased, and the yield of aquaculture is greatly improved.
In the culture medium, organic matters containing sodium ions are reduced as a carbon source, and phosphate buffer salt is added, so that the pH change of the strain is smooth in the culture process, and the growth of the strain is not influenced by the pH value of the strain exceeding 7.5.
The medium has reasonable carbon-nitrogen source proportion and comprehensive trace elements, and the rhodopseudomonas palustris has full nutrition, so that the pollution of algae and infectious microbes in the growth process can be effectively inhibited, and the problem which puzzles photosynthetic bacteria manufacturers for a long time is solved.
The rhodopseudomonas palustris produced by the culture medium formula has short growth period, the microbial inoculum has bright and uniform color and no wall adhesion and layering phenomenon, and the normal-temperature storage time can reach more than 1 year.
The rhodopseudomonas palustris microbial inoculum produced by the formula and specially used for aquaculture is applied to water plant aquaculture, so that the content of ammonia nitrogen and nitrite in water is quickly reduced, the dissolved oxygen in water is effectively increased, and the yield of aquaculture is greatly improved.
Drawings
FIG. 1 is OD in example 1 660nm A growth trend graph measured by the method;
FIG. 2 is a graph showing the growth tendency measured by the MPN5 method in example 1.
Detailed Description
The present invention will be described in further detail with reference to the following examples, but it should not be construed that the scope of the present invention is limited to the examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention, and the following embodiments are all completed by adopting the conventional prior art except for the specific description.
Example 1
A rhodopseudomonas palustris culture medium has the following formula: 1.05g/L of citric acid, 2.75g/L of beef extract, 0.52g/L of yeast extract, 1.93g/L of peptone, 0.50/L of sodium acetate, 0.35g/L of magnesium sulfate, 0.25g/L of calcium chloride, 0.14g/L of monopotassium phosphate, 5mg/L of ferrous sulfate, VB 6 0.2mg/L, 0.5mL/L of composite inorganic salt solution (EDTA 0.5g/L, zinc sulfate 0.01g/L, boric acid 0.03g/L, manganese chloride 0.003g/L, calcium chloride 0.02g/L, copper chloride 0.001g/L, sodium molybdate 0.003 g/L), pH6.8-7.0; sterilizing the potassium dihydrogen phosphate, the compound inorganic salt solution and the rest components in the culture medium at 115 deg.C for 10 min.
The culture medium is used as an experimental group, a control group is set, and the culture medium adopts a traditional RCVBN formula: 4.0g/L of DL-malic acid, 0.12g/L of magnesium sulfate, 1.0g/L of ammonium sulfate, 0.075g/L of calcium chloride, 0.5g/L of monopotassium phosphate, 0.3g/L of dipotassium hydrogen phosphate, 0.020g/L of EDTA-2Na, VB 1 0.001g/L, 0.001g/L nicotinic acid, 0.015g/L biotin and 1mL of T.M stock solution (0.7 g/L boric acid, 0.389g/L manganese sulfate, 0.188g/L sodium molybdate and 0.02g/L copper nitrate) and sterilizing by a conventional method.
The culture medium of the invention of example 1 and the control group culture medium are inoculated with the same strain and inoculum size under the same conditions and cultured, and the strain is Rhodopseudomonas palustris strain CGMCC 1.5007.
Specifically, two groups of culture media are inoculated with seed liquid in logarithmic phase, and the effective viable count is 6.20 x 10 9 Culturing in 20L plastic bucket with inoculum size of 10% and temperature of 33 deg.C, and illuminating with incandescent lamp at 4000Lux for 7 days. 2 methods are adopted to track and monitor the growth trend of rhodopseudomonas palustris.
(1) Tracking and detecting the absorbance value (namely OD) of 20 times diluted microbial inoculum under 660nm wavelength 660nm ) Drawing a growth curve (figure 1);
(2) The growth curves were plotted according to the MPN5 method of Standard NY527-2002 (FIG. 2).
As can be seen from the results of FIGS. 1 and 2, OD was found in the culture medium of the experimental group and the control group 660nm OD of the experimental group was substantially the same as the growth trend of the bacterial load, but at the same time period 660nm The culture medium is superior to the traditional RCVBN formula, is more beneficial to the rapid growth of the rhodopseudomonas palustris, and does not accelerate the aging of strains.
Example 2
A rhodopseudomonas palustris culture medium is prepared by the following steps (taking 20L of culture medium as an example):
(1) 22.0g of citric acid, 57.0g of beef extract, 12.0g of yeast extract, 39.0g of peptone, 11.0g of sodium acetate, 8.0g of magnesium sulfate, 160.0mg of ferrous sulfate and VB 6 4.0mg and 5.0g of calcium chloride are put in a beaker, the pH value is adjusted to 6.8 by 20 percent of potassium hydroxide, pure water is added to a constant volume of 19.9L, and the mixture is stirred to be uniformly mixed;
(2) Weighing 4.0g of monopotassium phosphate into a beaker, metering the volume of pure water to 100mL, and stirring to uniformly mix the pure water and the pure water;
(3) Weighing 0.5g of EDTA, 0.01g of zinc sulfate, 0.03g of boric acid, 0.003g of manganese chloride, 0.02g of calcium chloride, 0.001g of copper chloride and 0.003g of sodium molybdate in a beaker, fixing the volume of pure water to 1000mL, and stirring to uniformly mix the pure water and the sodium molybdate;
(5) Subpackaging the three solutions in steps (1), (2) and (3), sterilizing at 115 deg.C for 10 min.
(6) Cooling the sterilized solution to 60-80 ℃, mixing the two solutions (1) and (2) in an aseptic environment, adding 16mL of the solution (3), uniformly mixing, subpackaging sterilized culture containers such as test tubes, plastic bottles, plastic barrels and the like to obtain the prepared culture medium, cooling to room temperature, and carrying out inoculation.
The culture medium is used for preparing the rhodopseudomonas palustris microbial inoculum, the rhodopseudomonas palustris strain CGMCC1.5007 is selected, and the culture process is as follows:
(1) Activating strains: inoculating liquid strain in test tube (25mm. About.180mm) with good light transmittance, activating, inoculating at 40%, sealing, filling 90%, illuminating at 4000Lux, culturing at 33 deg.C for 4 days, and culturing during 4 daysThe test tube is rotated by shaking to make Rhodopseudomonas palustris fully receive illumination, and is beneficial to growth, after 4 days, the Rhodopseudomonas palustris obviously has special odor inherent in the bacteria agent, the color is gradually changed into reddish brown from light red, and the OD is 20 times diluted by pure water 660nm 0.289 and 5.70 × 10 bacteria content 9 one/mL.
(2) First-order seed culture: culturing the first-stage seeds by using a 500mL light-transmitting plastic bottle, sealing, culturing at constant temperature of 33 ℃, wherein the liquid loading amount is 90%, the illumination intensity is 4000Lux, the inoculation amount is 30%, the culturing time is 3 days, the test tube is rotated by shaking every day during the culturing period, so that the rhodopseudomonas palustris fully receives illumination, the growth is facilitated, the inherent special smell of the microbial inoculum is obviously generated after 3 days, the color is changed from light red to red gradually into reddish brown, and the OD is diluted by 20 times by using pure water 660nm Is 0.321, and the bacterial count is 6.30 x 10 9 One per mL.
(3) Secondary seed culture: culturing the second-stage seeds in 10L transparent plastic bucket, sealing, culturing at constant temperature of 33 deg.C with liquid loading amount of 90%, illumination intensity of 4000Lux, inoculation amount of 30%, culturing for 3 days, and color changed from light red, red to reddish brown to mature, diluting with pure water by 20 times, and culturing at constant temperature of 33 deg.C 660nm The bacterial count is 0.350 and 6.80 x 10 9 One per mL.
(4) Expanding production: the photosynthetic bacteria production adopts a 20L transparent plastic bucket, and the culture conditions are as follows: the illumination intensity of an incandescent lamp is 4000Lux, the temperature is controlled to be 33 ℃, the sealing is carried out, the liquid filling amount is 90 percent, the inoculation amount is 10 percent, the culture is carried out for 7 days, the inherent special smell of the microbial inoculum is obvious, the color is gradually changed from light red, red to red brown, the microbial inoculum is mature in culture, and OD (optical density) is 20 times that of pure water diluted 660nm It is 0.430, and the bacterial count is 8.49 × 10 9 one/mL.
The microbial inoculum is applied as follows:
south grass carp is cultivated in a fish pond of a certain farmer in Binzhou Boxing county, shandong province, the area of the fish pond is 5.0 mu, and the water depth is 2 meters. The special rhodopseudomonas palustris agent for aquaculture, which is subjected to amplification culture, is adopted to improve the water body of an aquaculture pond, and the weight ratio of the rhodopseudomonas palustris agent is 8: when the microbial inoculum is sprinkled for 00 days, the total amount is 10.0kg, after the microbial inoculum is used for two days, the ammonia nitrogen is reduced to 0.4mg/L from the original 1.6mg/L, the nitrite is reduced to below 0.01mg/L from 0.40mg/L, and the water body is clarified from turbidity, so that the water quality is greatly improved, the food intake of the fish is improved, and the yield of the fish is increased.
Example 3
A rhodopseudomonas palustris culture medium is prepared by the following steps (taking 20L of culture medium as an example):
(1) Weighing 18.0g of citric acid, 30.0g of beef extract, 10.0g of yeast extract, 36.0g of peptone, 10.0g of sodium acetate, 6.0g of magnesium sulfate, 100.0mg of ferrous sulfate and VB 6 3.0mg of calcium chloride and 4.0g of calcium chloride are put in a beaker, the pH value is adjusted to 6.8 by 20 percent of potassium hydroxide, pure water is added to a constant volume of 19.9L, and the mixture is stirred to be uniformly mixed;
(2) Weighing 2.0g of monopotassium phosphate into a beaker, metering the volume of pure water to 100mL, and stirring to uniformly mix the monopotassium phosphate and the pure water;
(3) Weighing 0.5g of EDTA, 0.01g of zinc sulfate, 0.03g of boric acid, 0.003g of manganese chloride, 0.02g of calcium chloride, 0.001g of copper chloride and 0.003g of sodium molybdate in a beaker, fixing the volume of pure water to 1000mL, and stirring to uniformly mix the pure water and the sodium molybdate;
(4) Subpackaging the three solutions obtained in the steps (1), (2) and (3), and sterilizing at 115 ℃ for 10 min.
(5) Cooling the sterilized solution to 60-80 ℃, mixing the two solutions (1) and (2) in an aseptic environment, adding 10mL (3), mixing uniformly, subpackaging sterilized culture containers such as test tubes, plastic bottles, plastic barrels and the like to obtain the prepared culture medium, cooling to room temperature, and inoculating.
The culture medium is used for preparing the rhodopseudomonas palustris microbial inoculum, and a rhodopseudomonas palustris strain CICC23812 is selected, and the culture process is as follows:
(1) Activating strains: inoculating liquid strain in a test tube (25mm. X.180mm) with good light transmittance for activation, wherein the inoculation amount is 40%, sealing, the liquid loading amount is 90%, the illumination intensity is 4000Lux, culturing at the constant temperature of 33 ℃, the culturing time is 4 days, the test tube is rotated by shaking every day during the culturing period, so that rhodopseudomonas palustris is fully illuminated, the growth is facilitated, the inherent special smell of the microbial agent is obviously present after 4 days, the color is changed from light red to red brown gradually, and the pure water is diluted by 20 times to OD (optical density) and is diluted by 20 times 660nm Is 0.230, and the bacterial count is 4.54 × 10 9 one/mL.
(2) First-stage seed culture: first levelSeeds are cultured by selecting a 500mL light-transmitting plastic bottle, sealing is carried out, the liquid loading amount is 90%, the illumination intensity is 4000Lux, the constant temperature culture is carried out at 33 ℃, the inoculation amount is 30%, the culture time is 3 days, a test tube is rotated by shaking every day during the culture period, the rhodopseudomonas palustris fully receives illumination, the growth is facilitated, the inherent special smell of the microbial inoculum is obviously generated after 3 days, the color is changed from light red to red gradually into reddish brown, and the OD is diluted by 20 times through pure water 660nm 0.260, the bacterial count is 5.13 x 10 9 one/mL.
(3) Secondary seed culture: culturing the second-stage seeds in 10L light-transmitting plastic bucket, sealing, culturing at constant temperature of 33 deg.C with liquid loading amount of 90%, illumination intensity of 4000Lux, inoculation amount of 30%, culturing time of 3 days, color of special smell inherent in the bacteria liquid gradually changed from light red, red to reddish brown to mature, diluting with pure water to 20 times OD 660nm Is 0.289, and has a bacterial content of 5.70 × 10 9 one/mL.
(4) Expanding production: the photosynthetic bacteria production adopts a 20L transparent plastic bucket, and the culture conditions are as follows: the illumination intensity of an incandescent lamp is 4000Lux, the temperature is controlled to be 33 ℃, the sealing is carried out, the liquid filling amount is 90 percent, the inoculation amount is 10 percent, the culture is carried out for 7 days, the inherent special smell of the microbial inoculum is obvious, the color is changed from light red, red to reddish brown gradually, the microbial inoculum is mature in culture, and OD (optical density) which is 20 times of that of pure water is diluted by pure water 660nm Is 0.415, and the bacterial count is 8.20 x 10 9 one/mL.
The microbial inoculum is applied as follows:
the pond area of a shrimp pond in a breeding greenhouse of a certain farmer in Boxing county, binzhou, shandong province is 7.0 mu, the water depth is 1.5m, photosynthetic bacteria are splashed from the larvae Z1 of the shrimps until the shrimps are discharged from the pond. The daily dosage is 1.0kg, and the stock solution is diluted and added after water is changed every morning. During the whole seedling raising period, the pH can be stabilized at about 8.0, the changes of ammonia nitrogen and COD are relatively smooth and are obviously lower than those of a control shrimp pond without using photosynthetic bacteria, the seedling raising amount of each cubic water body reaches 42.5 thousands of tails on average, and is improved by 120 percent compared with a control group; the survival rate of the prawn larva reaches 65 percent and is improved by 150 percent compared with the control group. Therefore, the microbial inoculum plays an important role in promoting the material circulation of the aquatic ecosystem and the water quality purification process.
Example 4
A rhodopseudomonas palustris culture medium is prepared by the following steps (taking 20L of culture medium as an example):
(1) Weighing 21.0g of citric acid, 55.0g of beef extract, 10.4g of yeast extract, 38.6g of peptone, 10.0g of sodium acetate, 7.0g of magnesium sulfate, 120.0mg of ferrous sulfate and VB 6 3.0mg of calcium chloride and 2.0g of calcium chloride are put in a beaker, the pH value is adjusted to 6.8 by 20 percent of potassium hydroxide, pure water is added to a constant volume of 19.9L, and the mixture is stirred to be uniformly mixed;
(2) Weighing 2.8g of monopotassium phosphate in a beaker, metering the volume of pure water to 100mL, and stirring to uniformly mix the pure water and the pure water;
(3) Weighing EDTA0.5g, zinc sulfate 0.01g, boric acid 0.03g, manganese chloride 0.003g, calcium chloride 0.02g, copper chloride 0.001g and sodium molybdate 0.003g in a beaker, fixing the volume of pure water to 1000mL, and stirring to uniformly mix the materials;
(4) Subpackaging the three solutions in steps (1), (2) and (3), sterilizing at 115 deg.C for 10 min.
(5) Cooling the sterilized solution to 60-80 ℃, mixing the two solutions (1) and (2) in an aseptic environment, adding 10mL (3), mixing uniformly, subpackaging sterilized culture containers such as test tubes, plastic bottles, plastic barrels and the like to obtain the prepared culture medium, cooling to room temperature, and inoculating.
The culture medium is used for preparing the rhodopseudomonas palustris microbial inoculum, and rhodopseudomonas palustris sold by chemical Limited of Jinan Okai is selected, wherein the culture process is as follows:
(1) Activating strains: inoculating liquid strain in a test tube (25mm. X.180mm) with good light transmittance for activation, wherein the inoculation amount is 40%, sealing, the liquid loading amount is 90%, the illumination intensity is 4000Lux, culturing at the constant temperature of 33 ℃, the culturing time is 4 days, the test tube is rotated by shaking every day during the culturing period, so that rhodopseudomonas palustris is fully illuminated, the growth is facilitated, the inherent special smell of the microbial agent is obviously present after 4 days, the color is changed from light red to red brown gradually, and the pure water is diluted by 20 times to OD (optical density) and is diluted by 20 times 660nm Is 0.298, and has a bacterial count of 5.88 × 10 9 one/mL.
(2) First-stage seed culture: culturing the first-stage seeds with 500mL transparent plastic bottle, sealing, culturing at constant temperature of 33 deg.C with liquid content of 90%, illumination intensity of 4000Lux, and inoculation amount of 30%, culturingRotating the test tube by shaking every day in the culture period for 3 days to make Rhodopseudomonas palustris fully receive illumination, and have special odor after 3 days, the color is changed from light red, red to red brown, and the OD is 20 times diluted with pure water 660nm It is 0.232, and the bacterial count is 6.37 x 10 9 one/mL.
(3) Secondary seed culture: culturing the second-stage seeds in 10L transparent plastic bucket, sealing, culturing at constant temperature of 33 deg.C with liquid loading amount of 90%, illumination intensity of 4000Lux, inoculation amount of 30%, culturing for 3 days, and color changed from light red, red to reddish brown to mature, diluting with pure water by 20 times, and culturing at constant temperature of 33 deg.C 660nm The bacterial count is 0.355 and 7.00 x 10 9 one/mL.
(4) Expanding production: the photosynthetic bacteria production adopts a 20L transparent plastic bucket, and the culture conditions are as follows: the illumination intensity of an incandescent lamp is 4000Lux, the temperature is controlled to be 33 ℃, the sealing is carried out, the liquid filling amount is 90 percent, the inoculation amount is 10 percent, the culture is carried out for 7 days, the inherent special smell of the microbial inoculum is obvious, the color is changed from light red, red to reddish brown gradually, the microbial inoculum is mature in culture, and OD (optical density) which is 20 times of that of pure water is diluted by pure water 660nm It is 0.457, and the bacterial amount is 8.96 x 10 9 One per mL.
The microbial inoculum is applied as follows:
the penaeus vannamei boone is cultured in a shrimp pond of a certain farmer in Lijin county of Shandong Ying City, the area of each shrimp pond is 7.5 mu, and the water depth is 2 meters. The water quality and water quality indexes before and after the microbial inoculum is used and the growth condition of the penaeus vannamei boone are shown in table 1. 2019.08.10 am 8:00 first sprinkling fungicide, 2019.08.25 am 8:00 the second time of sprinkling the microbial inoculum, and the application amount of the microbial inoculum is 15.0L each time. The other shrimp pond is used as a control and is not treated by the microbial inoculum, and other conditions are consistent.
TABLE 1 comparison table of effect before and after using of special rhodopseudomonas palustris agent for aquaculture
| Time | pH | Dissolved oxygen | Ammonia nitrogen (mg/L) | Sulfite (mg/L) |
| Before use | 7.6 | 5.5 | 0.90 | 0.60 |
| After use 2d | 8.3 | 8.2 | 0.30 | 0.10 |
| 30d after use | 8.3 | 8.5 | <0.10 | <0.10 |
As can be seen from the table 1, the content of ammonia nitrogen and nitrite in the pond is rapidly reduced after the rhodopseudomonas palustris microbial inoculum cultured by the culture medium disclosed by the invention is used in the aquaculture pond, the content of ammonia nitrogen is reduced to 0.30mg/L from 0.90mg/L, the content of nitrite is reduced to 0.10mg/L from 0.60mg/L, the content of dissolved oxygen in water is increased to 8.5 from 5.5, and the water environment is effectively improved. Meanwhile, the microbial inoculum is applied twice in one month, so that the penaeus vannamei grows rapidly, the average weight is increased from 25.0 g/tail to 52.6 g/tail, and the weight is increased by 110.4%; compared with a control group (the weight is increased by 80.4%) which is not treated by the microbial inoculum, the microbial inoculum is increased by 30 percent. Therefore, the product provided by the method can be used for an aquaculture pond, can effectively reduce ammonia nitrogen and sulfite in a water body, reduce toxicity, effectively improve the water body environment and improve the aquaculture yield, and is an excellent modifier for ecological aquaculture.
Claims (6)
1. A culture method of a rhodopseudomonas palustris microbial inoculum is characterized by comprising the following specific preparation steps of (1) strain activation, (2) strain culture, (3) enlarged culture:
(1) Activating strains: inoculating the strain in a light-transmitting test tube culture medium, activating, culturing at constant temperature of 30-50% and illumination intensity of 3000-5000Lux for 3-5 days under sealed condition at 30-33 deg.C, wherein the color is changed from light red, red to red brown gradually to obtain mature culture;
(2) Seed culture: transferring the activated strain obtained in the step (1) to a light-transmitting culture container for culture, wherein the inoculation amount is 30-50%, the illumination intensity is 3000-5000Lux, the constant-temperature sealed culture is carried out for 2-4 days at the temperature of 30-33 ℃, and the color is gradually changed from light red to red brown to obtain mature culture seed liquid;
(3) And (3) expanding culture: inoculating the seed liquid into a larger culture container, wherein the inoculation amount is 10-30%, and the culture conditions are as follows: the illumination intensity is 3000-5000Lux, the temperature is controlled at 30-33 ℃, and the culture is sealed for 5-7 days;
the adopted rhodopseudomonas palustris culture medium comprises the following components in percentage by weight: 0.75-1.25g/L of citric acid, 1.50-3.00g/L of beef extract, 0.42-0.62g/L of yeast extract, 1.50-2.00g/L of peptone, 0.40-0.60g/L of sodium acetate, 0.25-0.50g/L of magnesium sulfate, 5.00-8.00mg/L of ferrous sulfate and VB 6 0.15-0.2mg/L, 0.10-0.30g/L of calcium chloride, 0.10-0.25g/L of monopotassium phosphate, 0.5-1.0mL/L of composite inorganic salt solution, water as solvent, and adjusting the pH value to 6.8-7.0;
the composite inorganic salt solution comprises the following components: 0.5g/L of EDTA, 0.01g/L of zinc sulfate, 0.03g/L of boric acid, 0.003g/L of manganese chloride, 0.02g/L of calcium chloride, 0.001g/L of copper chloride, 0.003g/L of sodium molybdate and water as a solvent.
2. The method for culturing the rhodopseudomonas palustris microbial inoculum according to claim 1, wherein the pH of the rhodopseudomonas palustris culture medium is adjusted by using sodium hydroxide or potassium hydroxide with the mass concentration of 20%.
3. The method for culturing Rhodopseudomonas palustris microbial inoculum according to claim 1, wherein the potassium dihydrogen phosphate and the compound inorganic salt solution in the culture medium are sterilized separately.
4. The method for culturing the rhodopseudomonas palustris inoculum according to claim 1, wherein the tube is rotated by shaking at least 1 time every day during the strain activation culture in the step (1).
5. The method for culturing Rhodopseudomonas palustris microbial inoculum according to claim 1, wherein the total liquid loading of the test tube in the step (1) of strain activation is 90%.
6. The method for culturing rhodopseudomonas palustris inoculant according to claim 1, wherein all culture containers are subjected to chemical sterilization, specifically, 0.5 per mill of sodium hypochlorite aqueous solution is soaked for 12-24 hours or washed with 75% alcohol for 2 times; sterilizing potassium dihydrogen phosphate, compound inorganic salt solution, and other rest components at 115 deg.C for 10 min.
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