CN116445293A - Schizochytrium limacinum with efficient utilization of propionate, application and method for treating propionate wastewater and accumulating high-value DHA - Google Patents

Schizochytrium limacinum with efficient utilization of propionate, application and method for treating propionate wastewater and accumulating high-value DHA Download PDF

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CN116445293A
CN116445293A CN202310313325.4A CN202310313325A CN116445293A CN 116445293 A CN116445293 A CN 116445293A CN 202310313325 A CN202310313325 A CN 202310313325A CN 116445293 A CN116445293 A CN 116445293A
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propionate
sulfate
wastewater
culture medium
schizochytrium
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孙小曼
张自夷
马旺
郭东升
李颖风
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Nanjing Normal University
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Abstract

The invention discloses schizochytrium limacinum which efficiently utilizes propionate, uses the schizochytrium limacinum, processes propionate waste water and accumulates high-value DHA, wherein the schizochytrium limacinum is obtained by taking propionate waste water as a basis, preparing 2g/L, 4g/L, 6g/L and 8 g/L of propionate waste water by utilizing sodium propionate gradient, and preparing complete substitution culture medium by using the propionate waste water as a solvent for carrying out adaptive domestication on the schizochytrium limacinum. Based on schizochytrium culture, the invention adopts an adaptive laboratory evolution means to obtain the schizochytrium strain with high resistance to propionate wastewater, the strain can efficiently utilize propionate, and the obtained strain has the stability of multiple passages.

Description

Schizochytrium limacinum with efficient utilization of propionate, application and method for treating propionate wastewater and accumulating high-value DHA
Technical Field
The invention belongs to the technical field of biochemical engineering, and particularly relates to schizochytrium limacinum capable of efficiently utilizing propionate, application and a method for treating propionate wastewater and accumulating high-value DHA.
Background
Propionic acid (also known as primary oleic acid), which is a short chain saturated fatty acid of the formula CH 3 CH 2 COOH, which breaks down in the same way as the β -oxidation pathway of fatty acids, generally requires the formation of propionyl-CoA in combination with CoA for use by organisms. Since the body of propionyl-CoA contains 3 carbon atoms, it cannot directly participate in the β -oxidation process nor directly enter the tricarboxylic acid cycle. Propionic acid can change the permeability of the membrane, so that microorganisms are inactivated, and the microbial agent has good inhibition effect on mold and bacteria, so that propionic acid is often used as a preservative in the food processing process, and excessive propionic acid/propionate waste water is discharged due to the large-scale use. In addition, the conversion of coal gas into natural gas is a current popular trend for energy conservation and greenhouse gas emission control, and a wide coal industry layout is developed in China and European Union. However, 1 ton coal gasification produces 3 tons of wastewater, although the CO in the wastewater 2 ，H 2 Both S and phenolics have solutions, but propionic acid in wastewater lacks solutions.
The direct discharge of coal gasification wastewater not only can seriously pollute the environment, but also can cause great waste of water resources. Propionic acid is a weak electrolyte (ka=1.34×10) -5 ) The aqueous solution is weak acid and strong in corrosiveness, the steam has irritation to skin and respiratory tract, and the propionic acid wastewater has harm to environment and can pollute water; inhalation of propionic acid vapor has strong irritation to respiratory tract, can induce pulmonary edema of human body, and has strong irritation to human eyes.
The mass production and use of propionic acid/propionate can exacerbate its emissions to soil, water and other environments. At present, the domestic method for treating propionic acid wastewater mainly comprises the following steps: iron-carbon micro-electrolysis, coagulating sedimentation, UASB, secondary sedimentation tank treatment process, fenton method, flocculating sedimentation pretreatment, biochemical process and the like. Although the technology for treating propionic acid wastewater is gradually perfected, propionate in propionic acid wastewater is difficult to recycle. Although there are perfect even-numbered carbon metabolic pathways in microorganisms and animals, ingestion of propionate into the body produces propionyl-coa, and most microorganisms and animals cannot metabolize it, which can have serious effects on their growth and development.
Schizochytrium is a heterotrophic marine protist with high DHA content, and is widely applied to scientific research and commercial production due to the characteristics of high growth speed, rich DHA content, safety certification, easy culture and the like. Schizochytrium has a unique metabolic pathway to metabolize propionyl-coa, and is itself tolerant to propionate, and can maintain a high biomass at a given propionate concentration. Therefore, schizochytrium is selected for the utilization research of propionate, which is not only helpful for improving the environment, but also can produce various high-value products. However, the capacity of schizochytrium for the use of propionate has yet to be increased.
By searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provide a method for efficiently utilizing schizochytrium limacinum of propionate, applying and treating propionate wastewater and accumulating high-value DHA.
The technical scheme adopted for solving the technical problems is as follows:
schizochytrium with high efficiency of propionate is obtained by taking propionate waste water as a base, preparing 2g/L, 4g/L, 6g/L and 8g/L propionate waste water by utilizing sodium propionate gradient, and preparing complete substitution culture medium by using the propionate waste water as a solvent for adaptive domestication.
Further, the domestication method comprises the following steps:
adding 1ml of schizochytrium strain frozen in a 20% glycerol pipe into a seed culture medium of normal schizochytrium seed solution, namely clean water, serving as a seed culture medium of a solvent, continuously culturing for 3 generations, culturing for 24 hours in each generation, adding the schizochytrium strain into a seed culture medium of 2g/L propionate wastewater serving as the solvent according to an inoculum size ratio of 1%, domesticating for 30 generations, continuously adding the schizochytrium strain into a seed culture medium of 4g/L propionate wastewater serving as the solvent according to an inoculum size ratio of 1%, domesticating for 30 generations, continuously adding the schizochytrium strain into a seed culture medium of 6g/L propionate wastewater serving as the solvent according to an inoculum size ratio of 1%, domesticating for 30 generations, and continuously adding the schizochytrium strain into a seed culture medium of 8g/L propionate wastewater serving as the solvent according to an inoculum size ratio of 1%, domesticating for 30 generations; simultaneously, respectively taking 2g/L, 4g/L, 6g/L and 8g/L of seed liquid after the domestication of the propionate wastewater is finished, and respectively carrying out biomass measurement;
After successful domestication, schizochytrium limacinum with propionic acid tolerance capacity reaching 6g/L and efficient utilization of propionate is finally obtained.
Further, after the propionate concentration of the schizochytrium is more than 6g/L, the schizochytrium cannot grow;
or the propionate wastewater is from bakery production wastewater, sodium propionate is used as a preservative in the bread, and the wastewater contains sodium propionate with the concentration of 2.4g/L;
alternatively, the schizochytrium strain frozen in 20% glycerol is schizochytrium (schizochytrium sp.) HX-308 (this strain is a strain known in the art, and is disclosed, for example, in patent publication No. CN104974944a, which is preserved in the chinese collection of typical cultures (cctccc), with a preservation number of cctcccno. m 209059).
Further, the seed culture medium of the normal schizochytrium limacinum seed solution is: the pH of the medium was 6.4 and included: glucose 50g/L, yeast extract 5g/L, sodium sulfate 5g/L, magnesium sulfate 2g/L, ammonium sulfate 6g/L, potassium chloride 1g/L, calcium chloride 0.1g/L, potassium sulfate 0.6g/L, potassium dihydrogen phosphate 1g/L, sodium glutamate 10g/L, zinc sulfate heptahydrate 3mg/L, cobalt chloride hexahydrate 0.05mg/L, copper sulfate pentahydrate 5mg/L, nickel sulfate hexahydrate 1mg/L, ferric sulfate heptahydrate 10mg/L, calcium pantothenate 4mg/L, manganese chloride tetrahydrate 5mg/L, sodium molybdate dihydrate 0.04mg/L; the solvent is prepared by using clear water;
The culture medium taking 2g/L propionate wastewater as a solvent is as follows: the pH of the medium was 6.4 and included: glucose 50g/L, yeast extract 5g/L, sodium sulfate 5g/L, magnesium sulfate 2g/L, ammonium sulfate 6g/L, potassium chloride 1g/L, calcium chloride 0.1g/L, potassium sulfate 0.6g/L, potassium dihydrogen phosphate 1g/L, sodium glutamate 10g/L, zinc sulfate heptahydrate 3mg/L, cobalt chloride hexahydrate 0.05mg/L, copper sulfate pentahydrate 5mg/L, nickel sulfate hexahydrate 1mg/L, ferric sulfate heptahydrate 10mg/L, calcium pantothenate 4mg/L, manganese chloride tetrahydrate 5mg/L, sodium molybdate dihydrate 0.04mg/L; the solvent is prepared by using 2g/L propionate wastewater to completely replace clear water;
the culture medium taking 4g/L propionate wastewater as a solvent is as follows: the pH of the medium was 6.4 and included: glucose 50g/L, yeast extract 5g/L, sodium sulfate 5g/L, magnesium sulfate 2g/L, ammonium sulfate 6g/L, potassium chloride 1g/L, calcium chloride 0.1g/L, potassium sulfate 0.6g/L, potassium dihydrogen phosphate 1g/L, sodium glutamate 10g/L, zinc sulfate heptahydrate 3mg/L, cobalt chloride hexahydrate 0.05mg/L, copper sulfate pentahydrate 5mg/L, nickel sulfate hexahydrate 1mg/L, ferric sulfate heptahydrate 10mg/L, calcium pantothenate 4mg/L, manganese chloride tetrahydrate 5mg/L, sodium molybdate dihydrate 0.04mg/L; the solvent is prepared by using 4g/L propionate wastewater to completely replace clear water;
The culture medium taking 6g/L propionate wastewater as a solvent is as follows: the pH of the medium was 6.4 and included: glucose 50g/L, yeast extract 5g/L, sodium sulfate 5g/L, magnesium sulfate 2g/L, ammonium sulfate 6g/L, potassium chloride 1g/L, calcium chloride 0.1g/L, potassium sulfate 0.6g/L, potassium dihydrogen phosphate 1g/L, sodium glutamate 10g/L, zinc sulfate heptahydrate 3mg/L, cobalt chloride hexahydrate 0.05mg/L, copper sulfate pentahydrate 5mg/L, nickel sulfate hexahydrate 1mg/L, ferric sulfate heptahydrate 10mg/L, calcium pantothenate 4mg/L, manganese chloride tetrahydrate 5mg/L, sodium molybdate dihydrate 0.04mg/L; the solvent is prepared by using 6g/L propionate wastewater to completely replace clear water;
the culture medium taking 8g/L propionate wastewater as a solvent is as follows: the pH of the medium was 6.4 and included: glucose 50g/L, yeast extract 5g/L, sodium sulfate 5g/L, magnesium sulfate 2g/L, ammonium sulfate 6g/L, potassium chloride 1g/L, calcium chloride 0.1g/L, potassium sulfate 0.6g/L, potassium dihydrogen phosphate 1g/L, sodium glutamate 10g/L, zinc sulfate heptahydrate 3mg/L, cobalt chloride hexahydrate 0.05mg/L, copper sulfate pentahydrate 5mg/L, nickel sulfate hexahydrate 1mg/L, ferric sulfate heptahydrate 10mg/L, calcium pantothenate 4mg/L, manganese chloride tetrahydrate 5mg/L, sodium molybdate dihydrate 0.04mg/L; the solvent is prepared by using 8g/L propionate wastewater to completely replace clear water.
The application of schizochytrium in treating propionate wastewater and simultaneously preparing DHA by utilizing propionate efficiently as described above.
A method for treating propionate wastewater and accumulating high value DHA using schizochytrium efficiently utilizing propionate as described above, comprising the steps of:
inoculating schizochytrium limacinum strains with high efficiency by utilizing propionate into a seed culture medium for activation to obtain strains for fermentation; inoculating a strain for fermentation into a fermentation culture medium for fermentation culture; collecting thallus to extract grease to obtain DHA.
Further, the whole period of the fermentation culture can reach 60 hours, for example, 12 hours, 24 hours, 36 hours, 48 hours or 60 hours;
or the culture condition is 25-30 ℃ and 150-250 r/min shaking culture;
alternatively, the method for collecting the thalli and extracting the grease comprises the following steps:
1) Adding NaOH solution into fermentation broth after fermentation culture is finished to adjust pH=11-13, adding wall breaking enzyme with the mass final concentration of 0.01-0.4%, and oscillating for 5-15 h at 40-60 ℃ at 100-200 r/min;
2) Cooling to room temperature, and adding equal volume of absolute ethyl alcohol to inactivate wall-breaking enzyme;
3) Extracting with n-hexane, and collecting the upper organic phase;
4) Repeating step 3) for several times, mixing the organic phases, volatilizing the solvent to obtain lipid
Further, the seed culture medium and the fermentation culture medium are prepared by completely using propionate wastewater as a solvent;
alternatively, the strain for fermentation is obtained by the following method:
inoculating schizochytrium limacinum strain with high efficiency by using propionate into a flat-plate culture medium for culture, picking single bacterial colony, inoculating into a seed culture medium for activation, and culturing to obtain first-stage seeds; inoculating the first-level seeds into a seed culture medium, and culturing to obtain second-level seeds; inoculating the secondary seeds into a seed culture medium, and culturing to obtain tertiary seeds serving as strains for fermentation.
Further, the plate medium is: the pH value of the culture medium is 6.0-6.5, and the culture medium comprises: 15-20g/L of agar, 30-60g/L of glucose, 8-15 g/L of yeast extract powder, 10-15 g/L of sodium sulfate, 2-4 g/L of magnesium sulfate, 6-12 g/L of ammonium sulfate, 1-2 g/L of potassium chloride, 0.1-0.2 g/L of calcium chloride, 0.5-1 g/L of potassium sulfate, 0.5-2 g/L of monopotassium phosphate, 8-12 g/L of sodium glutamate, 1-5 mg/L of zinc sulfate heptahydrate, 0.01-0.1 g/L of cobalt chloride hexahydrate, 2-6 mg/L of copper sulfate pentahydrate, 1-2 mg/L of nickel sulfate hexahydrate, 8-15 mg/L of ferric sulfate heptahydrate, 2-4 mg/L of calcium pantothenate, 3-5 mg/L of manganese chloride tetrahydrate, 0.04mg/L of sodium molybdate dihydrate and vitamin B 6 3-10mg/L, vitamin B 12 0.3-1.2mg/L; the solvent is prepared by using 6g/L propionate wastewater to completely replace clear water;
the seed culture medium is as follows: the pH value of the culture medium is 6.0-6.5, and the culture medium comprises: 40-60g/L of glucose, 4-6 g/L of yeast extract powder, 5-8 g/L of sodium sulfate, 2-4 g/L of magnesium sulfate, 4-8 g/L of ammonium sulfate, 1-2 g/L of potassium chloride, 0.1-0.2 g/L of calcium chloride, 0.5-1 g/L of potassium sulfate, 0.5-2 g/L of monopotassium phosphate, 8-12 g/L of sodium glutamate, 1-5 mg/L of zinc sulfate heptahydrate, 0.01-0.1 mg/L of cobalt chloride hexahydrate, 2-6 mg/L of copper sulfate pentahydrate, 1-2 mg/L of nickel sulfate hexahydrate, 8-15 mg/L of ferric sulfate heptahydrate, 2-4 mg/L of calcium pantothenate, 3-5 mg/L of manganese chloride tetrahydrate and 0.04mg/L of sodium molybdate dihydrate; the solvent is prepared by using 6g/L propionate wastewater to completely replace clear water;
the fermentation medium is as follows: the pH value of the culture medium is 6.0-6.5, and the culture medium comprises: 60-100g/L glucose, 5-15 g/L yeast extract powder, 5-12 g/L sodium sulfate, 2-4 g/L magnesium sulfate, 4-8 g/L ammonium sulfate, 1-2 g/L potassium chloride, 0.1-0.2 g/L calcium chloride, 0.5-1 g/L potassium sulfate, 0.5-2 g/L potassium dihydrogen phosphate, 15-20 g/L sodium glutamate, 1-5 mg/L zinc sulfate heptahydrate, 0.01-0.1 mg/L cobalt chloride hexahydrate, 2-6 mg/L copper sulfate pentahydrate, 1-2 mg/L nickel sulfate hexahydrate, 8-15 mg/L ferric sulfate heptahydrate, 2-4 mg/L calcium pantothenate, 3-5 mg/L manganese chloride tetrahydrate, 0.04mg/L sodium molybdate dihydrate, vitamins B 6 3-10mg/L, vitamin B 12 0.1-0.8mg/L; the solvent is prepared by using 6g/L propionate wastewater to completely replace clean water.
A method for accumulating high-value DHA and biomass by fermenting schizochytrium by utilizing schizochytrium which efficiently utilizes propionate and replacing culture medium with completely propionate wastewater, which is characterized in that: the method comprises the following steps:
inoculating schizochytrium limacinum which efficiently utilizes propionate into a seed culture medium with 6g/L sodium propionate wastewater completely replaced according to a proportion of 1%, culturing at 28 ℃ and 170rpm for 24 hours, namely, continuously culturing for 3 generations; inoculating 10% of the mixed solution into fermentation liquid for fermentation, wherein the initial rotation speed is 300rpm, the ventilation amount is 2ppm, the temperature is kept at 28 ℃, and the dissolved oxygen is 15% -30%; controlling the pH value to be 6.5-7.0, performing pH regulation by using pre-prepared acid and alkali, sampling and measuring the glucose concentration every 12 hours, keeping the glucose concentration in a fermentation tank to be above 30g/L, and continuously culturing for 120 hours;
the acid, the alkali and the sugar used in the method are also prepared by using 6g/L propionic acid wastewater as a solvent, wherein the acid is citric acid monohydrate, and the alkali is sodium hydroxide;
the formula of the fermentation liquor is as follows: the pH value is 6.0-6.5, and comprises: 60-100g/L of glucose, 5-15 g/L of yeast extract powder, 5-12 g/L of sodium sulfate, 2-4 g/L of magnesium sulfate, 4-8 g/L of ammonium sulfate, 1-2 g/L of potassium chloride, 0.1-0.2 g/L of calcium chloride, 0.5-1 g/L of potassium sulfate, 0.5-2 g/L of monopotassium phosphate, 15-20 g/L of sodium glutamate, 1-5 mg/L of zinc sulfate heptahydrate, 0.01-0.1 mg/L of cobalt chloride hexahydrate, 2-6 mg/L of copper sulfate pentahydrate, 1-2 mg/L of nickel sulfate hexahydrate, 8-15 mg/L of ferric sulfate heptahydrate, 2-4 mg/L of calcium pantothenate, 3-5 mg/L of manganese chloride tetrahydrate, 0.04mg/L of sodium molybdate dihydrate, 63-10mg/L of vitamin B 12 0.1-0.8mg/L, the solvent is prepared by using 6g/L propionate waste water to completely replace clean water, sterilizing for 30 minutes at 115 ℃, and cooling.
The beneficial effects obtained by the invention are as follows:
1. based on schizochytrium culture, the invention adopts an adaptive laboratory evolution means to obtain the schizochytrium strain with high tolerance to propionate wastewater, the strain can efficiently utilize propionate, and the obtained strain can simultaneously accumulate biomass and produce DHA in a culture medium taking the propionate wastewater as a solvent.
2. The schizochytrium strain obtained by the invention is cultured in a complete propionate wastewater culture medium, and the oxidation resistance and carotenoid accumulation capacity of the schizochytrium are obviously enhanced.
3. The invention uses propionate waste water to completely prepare the culture medium, and the biomass accumulation and DHA synthesis capacity of schizochytrium limacinum are obviously enhanced, which provides a theoretical basis for industrial metabolism of the propionate waste water by the strain.
4. The invention relates to a schizochytrium limacinum obtaining method for decomposing and utilizing propionic acid wastewater. The method has important significance for improving environment and developing products by utilizing propionic acid wastewater in the future schizochytrium.
5. The schizochytrium limacinum strain with high tolerance to propionate is obtained adaptively. The propionate concentration in the bakery wastewater is 2.4g/L, but the propionate concentration in the coal gasification wastewater is very high and can partially reach 10g/L. Therefore, 2,4,6,8g/L propionate wastewater completely replaces a culture medium to carry out adaptive domestication on schizochytrium, schizochytrium (schizochytriumsp.CCTCCM 209759) is adopted as an original strain, and a schizochytrium strain capable of efficiently catabolizing propionic acid is obtained through an adaptive laboratory evolution means, so that the method has important significance for improving environment and developing products by utilizing propionic acid wastewater for microbial metabolism.
Drawings
FIG. 1 is a graph showing the comparison of growth of schizochytrium HX-308 of example 1 according to the invention at various sodium propionate concentrations;
FIG. 2 is a diagram showing the metabolic propionic acid process of schizochytrium in example 1 of the present invention;
FIG. 3 is a graph showing the domestication result of schizochytrium in the graded concentration propionate wastewater complete replacement medium in example 2 of the present invention;
FIG. 4 is a comparative graph of lipid composition obtained after fermentation of the domesticated schizochytrium strain of example 3 of the present invention;
FIG. 5 is a graph showing the comparison of carotenoid production obtained after fermentation of the domesticated schizochytrium strain of example 3 of the present invention;
FIG. 6 is a graph showing the results of feed fermentation production of domesticated schizochytrium in propionic acid wastewater complete replacement medium in example 4 of the present invention.
Detailed Description
The present invention will be further described in detail with reference to examples, but the scope of the present invention is not limited to the examples.
The raw materials used in the invention are conventional commercial products unless specified otherwise, the methods used in the invention are conventional methods in the art unless specified otherwise, and the mass of each substance used in the invention is conventional.
Schizochytrium with high efficiency of propionate is obtained by taking propionate waste water as a base, preparing 2g/L, 4g/L, 6g/L and 8g/L propionate waste water by utilizing sodium propionate gradient, and preparing complete substitution culture medium by using the propionate waste water as a solvent for adaptive domestication.
Preferably, the method of acclimatizing comprises the steps of:
adding 1ml of schizochytrium strain frozen in a 20% glycerol pipe into a seed culture medium of normal schizochytrium seed solution, namely clean water, serving as a seed culture medium of a solvent, continuously culturing for 3 generations, culturing for 24 hours in each generation, adding the schizochytrium strain into a seed culture medium of 2g/L propionate wastewater serving as the solvent according to an inoculum size ratio of 1%, domesticating for 30 generations, continuously adding the schizochytrium strain into a seed culture medium of 4g/L propionate wastewater serving as the solvent according to an inoculum size ratio of 1%, domesticating for 30 generations, continuously adding the schizochytrium strain into a seed culture medium of 6g/L propionate wastewater serving as the solvent according to an inoculum size ratio of 1%, domesticating for 30 generations, and continuously adding the schizochytrium strain into a seed culture medium of 8g/L propionate wastewater serving as the solvent according to an inoculum size ratio of 1%, domesticating for 30 generations; simultaneously, respectively taking 2g/L, 4g/L, 6g/L and 8g/L of seed liquid after the domestication of the propionate wastewater is finished, and respectively carrying out biomass measurement;
After successful domestication, schizochytrium limacinum with propionic acid tolerance capacity reaching 6g/L and efficient utilization of propionate is finally obtained.
Preferably, the schizochytrium is incapable of growing after a propionate concentration of more than 6 g/L;
or the propionate wastewater is from bakery production wastewater, sodium propionate is used as a preservative in the bread, and the wastewater contains sodium propionate with the concentration of 2.4g/L;
alternatively, the schizochytrium strain frozen in 20% glycerol is schizochytrium (schizochytrium sp.) HX-308 (this strain is a strain known in the art, and is disclosed, for example, in patent publication No. CN104974944a, which is preserved in the China Center for Type Culture Collection (CCTCC), with a preservation number of cctccc No. m 209059).
Further, the seed culture medium of the normal schizochytrium limacinum seed solution is: the pH of the medium was 6.4 and included: glucose 50g/L, yeast extract 5g/L, sodium sulfate 5g/L, magnesium sulfate 2g/L, ammonium sulfate 6g/L, potassium chloride 1g/L, calcium chloride 0.1g/L, potassium sulfate 0.6g/L, potassium dihydrogen phosphate 1g/L, sodium glutamate 10g/L, zinc sulfate heptahydrate 3mg/L, cobalt chloride hexahydrate 0.05mg/L, copper sulfate pentahydrate 5mg/L, nickel sulfate hexahydrate 1mg/L, ferric sulfate heptahydrate 10mg/L, calcium pantothenate 4mg/L, manganese chloride tetrahydrate 5mg/L, sodium molybdate dihydrate 0.04mg/L; the solvent is prepared by using clear water;
The culture medium taking 2g/L propionate wastewater as a solvent is as follows: the pH of the medium was 6.4 and included: glucose 50g/L, yeast extract 5g/L, sodium sulfate 5g/L, magnesium sulfate 2g/L, ammonium sulfate 6g/L, potassium chloride 1g/L, calcium chloride 0.1g/L, potassium sulfate 0.6g/L, potassium dihydrogen phosphate 1g/L, sodium glutamate 10g/L, zinc sulfate heptahydrate 3mg/L, cobalt chloride hexahydrate 0.05mg/L, copper sulfate pentahydrate 5mg/L, nickel sulfate hexahydrate 1mg/L, ferric sulfate heptahydrate 10mg/L, calcium pantothenate 4mg/L, manganese chloride tetrahydrate 5mg/L, sodium molybdate dihydrate 0.04mg/L; the solvent is prepared by using 2g/L propionate wastewater to completely replace clear water;
the culture medium taking 4g/L propionate wastewater as a solvent is as follows: the pH of the medium was 6.4 and included: glucose 50g/L, yeast extract 5g/L, sodium sulfate 5g/L, magnesium sulfate 2g/L, ammonium sulfate 6g/L, potassium chloride 1g/L, calcium chloride 0.1g/L, potassium sulfate 0.6g/L, potassium dihydrogen phosphate 1g/L, sodium glutamate 10g/L, zinc sulfate heptahydrate 3mg/L, cobalt chloride hexahydrate 0.05mg/L, copper sulfate pentahydrate 5mg/L, nickel sulfate hexahydrate 1mg/L, ferric sulfate heptahydrate 10mg/L, calcium pantothenate 4mg/L, manganese chloride tetrahydrate 5mg/L, sodium molybdate dihydrate 0.04mg/L; the solvent is prepared by using 4g/L propionate wastewater to completely replace clear water;
The culture medium taking 6g/L propionate wastewater as a solvent is as follows: the pH of the medium was 6.4 and included: glucose 50g/L, yeast extract 5g/L, sodium sulfate 5g/L, magnesium sulfate 2g/L, ammonium sulfate 6g/L, potassium chloride 1g/L, calcium chloride 0.1g/L, potassium sulfate 0.6g/L, potassium dihydrogen phosphate 1g/L, sodium glutamate 10g/L, zinc sulfate heptahydrate 3mg/L, cobalt chloride hexahydrate 0.05mg/L, copper sulfate pentahydrate 5mg/L, nickel sulfate hexahydrate 1mg/L, ferric sulfate heptahydrate 10mg/L, calcium pantothenate 4mg/L, manganese chloride tetrahydrate 5mg/L, sodium molybdate dihydrate 0.04mg/L; the solvent is prepared by using 6g/L propionate wastewater to completely replace clear water;
the culture medium taking 8g/L propionate wastewater as a solvent is as follows: the pH of the medium was 6.4 and included: glucose 50g/L, yeast extract 5g/L, sodium sulfate 5g/L, magnesium sulfate 2g/L, ammonium sulfate 6g/L, potassium chloride 1g/L, calcium chloride 0.1g/L, potassium sulfate 0.6g/L, potassium dihydrogen phosphate 1g/L, sodium glutamate 10g/L, zinc sulfate heptahydrate 3mg/L, cobalt chloride hexahydrate 0.05mg/L, copper sulfate pentahydrate 5mg/L, nickel sulfate hexahydrate 1mg/L, ferric sulfate heptahydrate 10mg/L, calcium pantothenate 4mg/L, manganese chloride tetrahydrate 5mg/L, sodium molybdate dihydrate 0.04mg/L; the solvent is prepared by using 8g/L propionate wastewater to completely replace clear water.
The application of schizochytrium in treating propionate wastewater and simultaneously preparing DHA by utilizing propionate efficiently as described above.
A method for treating propionate wastewater and accumulating high value DHA using schizochytrium efficiently utilizing propionate as described above, comprising the steps of:
inoculating schizochytrium limacinum strains with high efficiency by utilizing propionate into a seed culture medium for activation to obtain strains for fermentation; inoculating a strain for fermentation into a fermentation culture medium for fermentation culture; collecting thallus to extract grease to obtain DHA.
Preferably, the whole period of the fermentation culture can reach 60 hours, for example, 12 hours, 24 hours, 36 hours, 48 hours or 60 hours;
or the culture condition is 25-30 ℃ and 150-250 r/min shaking culture;
alternatively, the method for collecting the thalli and extracting the grease comprises the following steps:
1) Adding NaOH solution into fermentation broth after fermentation culture is finished to adjust pH=11-13, adding wall breaking enzyme with the mass final concentration of 0.01-0.4%, and oscillating for 5-15 h at 40-60 ℃ at 100-200 r/min;
2) Cooling to room temperature, and adding equal volume of absolute ethyl alcohol to inactivate wall-breaking enzyme;
3) Extracting with n-hexane, and collecting the upper organic phase;
4) Repeating the step 3) for several times, combining the organic phases, volatilizing the solvent to obtain lipid;
Preferably, the seed culture medium and the fermentation culture medium are prepared by completely using propionate wastewater as a solvent;
alternatively, the strain for fermentation is obtained by the following method:
inoculating schizochytrium limacinum strain with high efficiency by using propionate into a flat-plate culture medium for culture, picking single bacterial colony, inoculating into a seed culture medium for activation, and culturing to obtain first-stage seeds; inoculating the first-level seeds into a seed culture medium, and culturing to obtain second-level seeds; inoculating the secondary seeds into a seed culture medium, and culturing to obtain tertiary seeds serving as strains for fermentation.
Preferably, the plate medium is: the pH value of the culture medium is 6.0-6.5, and the culture medium comprises: 15-20g/L of agar, 30-60g/L of glucose, 8-15 g/L of yeast extract powder, 10-15 g/L of sodium sulfate, 2-4 g/L of magnesium sulfate, 6-12 g/L of ammonium sulfate, 1-2 g/L of potassium chloride, 0.1-0.2 g/L of calcium chloride, 0.5-1 g/L of potassium sulfate, 0.5-2 g/L of monopotassium phosphate, 8-12 g/L of sodium glutamate, 1-5 mg/L of zinc sulfate heptahydrate, 0.01-0.1 g/L of cobalt chloride hexahydrate, 2-6 mg/L of copper sulfate pentahydrate, 1-2 mg/L of nickel sulfate hexahydrate, 8-15 mg/L of ferric sulfate heptahydrate, 2-4 mg/L of calcium pantothenate, 3-5 mg/L of manganese chloride tetrahydrate, 0.04mg/L of sodium molybdate dihydrate and vitamin B 6 3-10mg/L, vitamin B 12 0.3-1.2mg/L; the solvent is prepared by using 6g/L propionate wastewater to completely replace clear water;
the seed culture medium is as follows: the pH value of the culture medium is 6.0-6.5, and the culture medium comprises: 40-60g/L of glucose, 4-6 g/L of yeast extract powder, 5-8 g/L of sodium sulfate, 2-4 g/L of magnesium sulfate, 4-8 g/L of ammonium sulfate, 1-2 g/L of potassium chloride, 0.1-0.2 g/L of calcium chloride, 0.5-1 g/L of potassium sulfate, 0.5-2 g/L of monopotassium phosphate, 8-12 g/L of sodium glutamate, 1-5 mg/L of zinc sulfate heptahydrate, 0.01-0.1 mg/L of cobalt chloride hexahydrate, 2-6 mg/L of copper sulfate pentahydrate, 1-2 mg/L of nickel sulfate hexahydrate, 8-15 mg/L of ferric sulfate heptahydrate, 2-4 mg/L of calcium pantothenate, 3-5 mg/L of manganese chloride tetrahydrate and 0.04mg/L of sodium molybdate dihydrate; the solvent is prepared by using 6g/L propionate wastewater to completely replace clear water;
the fermentation medium is as follows: the pH value of the culture medium is 6.0-6.5, and the culture medium comprises: 60-100g/L glucose, 5-15 g/L yeast extract powder, 5-12 g/L sodium sulfate, 2-4 g/L magnesium sulfate, 4-8 g/L ammonium sulfate, 1-2 g/L potassium chloride, 0.1-0.2 g/L calcium chloride, 0.5-1 g/L potassium sulfate, 0.5-2 g/L potassium dihydrogen phosphate, 15-20 g/L sodium glutamate, 1-5 mg/L zinc sulfate heptahydrate, 0.01-0.1 mg/L cobalt chloride hexahydrate, 2-6 mg/L copper sulfate pentahydrate, 1-2 mg/L nickel sulfate hexahydrate, 8-15 mg/L ferric sulfate heptahydrate, 2-4 mg/L calcium pantothenate, 3-5 mg/L manganese chloride tetrahydrate, 0.04mg/L sodium molybdate dihydrate, and vitamin B 6 3-10mg/L, vitamin B 12 0.1-0.8mg/L; the solvent is prepared by using 6g/L propionate wastewater to completely replace clean water.
A method for accumulating high-value DHA and biomass by fermenting schizochytrium by utilizing schizochytrium which efficiently utilizes propionate and replacing culture medium with completely propionate wastewater, which is characterized in that: the method comprises the following steps:
inoculating schizochytrium limacinum which efficiently utilizes propionate into a seed culture medium with 6g/L sodium propionate wastewater completely replaced according to a proportion of 1%, culturing at 28 ℃ and 170rpm for 24 hours, namely, continuously culturing for 3 generations; inoculating 10% of the mixed solution into fermentation liquid for fermentation, wherein the initial rotation speed is 300rpm, the ventilation amount is 2ppm, the temperature is kept at 28 ℃, and the dissolved oxygen is 15% -30%; controlling the pH value to be 6.5-7.0, performing pH regulation by using pre-prepared acid and alkali, sampling and measuring the glucose concentration every 12 hours, keeping the glucose concentration in a fermentation tank to be above 30g/L, and continuously culturing for 120 hours;
the acid, the alkali and the sugar used in the method are also prepared by using 6g/L propionic acid wastewater as a solvent, wherein the acid is citric acid monohydrate, and the alkali is sodium hydroxide;
the formula of the fermentation liquor is as follows: the pH value is 6.0-6.5, and comprises: 60-100g/L of glucose, 5-15 g/L of yeast extract powder, 5-12 g/L of sodium sulfate, 2-4 g/L of magnesium sulfate, 4-8 g/L of ammonium sulfate, 1-2 g/L of potassium chloride, 0.1-0.2 g/L of calcium chloride, 0.5-1 g/L of potassium sulfate, 0.5-2 g/L of monopotassium phosphate, 15-20 g/L of sodium glutamate, 1-5 mg/L of zinc sulfate heptahydrate, 0.01-0.1 mg/L of cobalt chloride hexahydrate, 2-6 mg/L of copper sulfate pentahydrate, 1-2 mg/L of nickel sulfate hexahydrate, 8-15 mg/L of ferric sulfate heptahydrate, 2-4 mg/L of calcium pantothenate, 3-5 mg/L of manganese chloride tetrahydrate, 0.04mg/L of sodium molybdate dihydrate, 63-10mg/L of vitamin B 12 0.1-0.8mg/L, the solvent is prepared by using 6g/L propionate waste water to completely replace clean water, sterilizing for 30 minutes at 115 ℃, and cooling.
Specifically, the preparation and detection of the correlation are as follows:
the equipment, reagents, processes, parameters, etc. according to the present invention are conventional equipment, reagents, processes, parameters, etc. unless otherwise specified, and are not exemplified.
All ranges recited herein are inclusive of all point values within the range.
In the invention, except for the general meaning or special description in the field, the percentages are mass percentages, and the proportions are mass ratios. The unit of mass is, for example, gram, kilogram or ton.
In the present invention, the "room temperature" is a conventional ambient temperature, and may be 10 to 30 ℃.
The following media were used in the examples:
the pH of the plate medium was 6.4 and the medium consisted of and included: 15-20g/L of agar, 40g/L of glucose, 10g/L of yeast extract powder, 10g/L of sodium sulfate, 2g/L of magnesium sulfate, 6g/L of ammonium sulfate, 1g/L of potassium chloride, 0.1g/L of calcium chloride, 0.6g/L of potassium sulfate, 1g/L of monopotassium phosphate, 10g/L of sodium glutamate, 3mg/L of zinc sulfate heptahydrate, 0.05mg/L of cobalt chloride hexahydrate, 5mg/L of copper sulfate pentahydrate, 1mg/L of nickel sulfate hexahydrate, 10mg/L of ferric sulfate heptahydrate, 4mg/L of calcium pantothenate, 5mg/L of manganese chloride tetrahydrate, 0.04mg/L of sodium molybdate dihydrate, 16mg/L of vitamin B and 120.5mg/L of vitamin B. The solvent is prepared by completely replacing propionate waste water.
The pH of the seed medium of the seed solution was 6.4 and included: glucose 50g/L, yeast extract 5g/L, sodium sulfate 5g/L, magnesium sulfate 2g/L, ammonium sulfate 6g/L, potassium chloride 1g/L, calcium chloride 0.1g/L, potassium sulfate 0.6g/L, potassium dihydrogen phosphate 1g/L, sodium glutamate 10g/L, zinc sulfate heptahydrate 3mg/L, cobalt chloride hexahydrate 0.05mg/L, copper sulfate pentahydrate 5mg/L, nickel sulfate hexahydrate 1mg/L, ferric sulfate heptahydrate 10mg/L, calcium pantothenate 4mg/L, manganese chloride tetrahydrate 5mg/L, and sodium molybdate dihydrate 0.04mg/L. The solvent is prepared by completely replacing propionate waste water.
Wherein the pH value of the fermentation culture medium is 6.0-6.5, and the fermentation culture medium comprises: 80g/L of glucose, 10g/L of yeast extract powder, 10g/L of sodium sulfate, 2g/L of magnesium sulfate, 6g/L of ammonium sulfate, 1g/L of potassium chloride, 0.1g/L of calcium chloride, 0.6g/L of potassium sulfate, 1g/L of monopotassium phosphate, 10g/L of sodium glutamate, 3mg/L of zinc sulfate heptahydrate, 0.05mg/L of cobalt chloride hexahydrate, 5mg/L of copper sulfate pentahydrate, 1mg/L of nickel sulfate hexahydrate, 10mg/L of ferric sulfate heptahydrate, 4mg/L of calcium pantothenate, 5mg/L of manganese chloride tetrahydrate, 0.04mg/L of sodium molybdate dihydrate, 15mg/L of vitamin B and 120.5mg/L of vitamin B. The solvent is prepared by completely replacing propionate waste water.
In the feed fermentation, the acid liquor is prepared by citric acid monohydrate, and the concentration is 150g/L; the alkali liquor is prepared by sodium hydroxide with the concentration of 50g/L; the preparation concentration of the sugar supplementing liquid is 800g/L. The propionate wastewater is used for completely replacing clear water to be prepared as a solvent.
Example 1 evaluation of the ability of schizochytrium to metabolize propionate waste Water
Using the obtained propionate waste water, carrying out gradient preparation by utilizing sodium propionate and clear water, carrying out fermentation culture on schizochytrium at the concentration of sodium propionate of 0.5g/L, 1.0g/L, 1.5g/L, 2.0g/L, 2.5g/L, 3.0g/L, 3.5g/L and 4.0g/L, and detecting grease and biomass of schizochytrium.
In particular, the components and the addition amount of the seed liquid are as above seed culture medium, but the solvent is divided into clear water (0 g/L sodium propionate), 0.5g/L, 1.0g/L, 1.5g/L, 2.0g/L, 2.5g/L, 3.0g/L, 3.5g/L, 4.0g/L sodium propionate wastewater, and 9 seed liquids in total. The components and the addition amount of the fermentation broth are as above, but the solvent is divided into clear water (0 g/L sodium propionate), 0.5g/L, 1.0g/L, 1.5g/L, 2.0g/L, 2.5g/L, 3.0g/L, 3.5g/L and 4.0g/L sodium propionate wastewater, and the total amount of the fermentation broth is 9.
The fermentation process is as follows, the frozen schizochytrium is taken out from the freezing tube, added into the 9 seed solutions according to the proportion of 1 percent, cultured for 24 hours at 28 ℃ and 170rpm, and is called first generation, and continuously cultured for 3 generations. Respectively adding the above 9 fermentation broths according to the proportion of 10%, culturing at 28 ℃ and 170rpm for 36h, measuring biomass and lipid, and analyzing the potential of sodium propionate wastewater to replace clear water.
The method for measuring the biomass is as follows:
(1) 10ml of the fermentation broth was taken and filtered using a weighed filter paper.
(2) And after the liquid in the seed liquid is filtered, putting the filter paper into a 60-DEG oven for drying for 48 hours.
(3) After the filter paper dry weight no longer changed, the biomass was weighed and calculated.
The method for extracting lipid from the collected thalli comprises the following steps:
(1) Adding NaOH solution into fermentation broth after fermentation culture is finished to adjust pH to 10-13, adding 0.01-0.2% wall breaking enzyme, oscillating for 5-15 h at 40-60 ℃ at 100-200 r/min;
(2) Cooling to room temperature, and adding equal volume of absolute ethyl alcohol to inactivate wall-breaking enzyme;
(3) Extracting with n-hexane, and collecting the upper organic phase;
(4) Removing n-hexane by rotary evaporation, and weighing the lipid.
The results are shown in figure 1, and show that schizochytrium can maintain higher biological activity and metabolic capacity under the condition of a certain concentration of sodium propionate, wherein grease production and biomass of schizochytrium are not affected under the condition of a concentration of 1.5g/L sodium propionate. After a propionate concentration of more than 1.5g/L, the biomass of schizochytrium began to decline and schizochytrium did not grow substantially at a sodium propionate concentration of 3 g/L.
As shown in FIG. 2, schizochytrium was found to have three metabolic pathways to metabolize propionic acid wastewater. Thus, schizochytrium has the potential to continue to increase propionate wastewater.
Example 2 adaptive evolutionary schizochytrium tolerating high concentration propionate wastewater
Based on the obtained propionate wastewater, preparing 2,4,6,8g/L propionate wastewater by utilizing sodium propionate gradient, and preparing a complete substitution culture medium by using the propionate wastewater as a solvent to carry out adaptive domestication on schizochytrium.
The domestication steps are as follows: adding 1ml of schizochytrium strain frozen in a 20% glycerol pipe into a seed culture medium of normal schizochytrium seed solution (namely clean water is used as a solvent), continuously culturing for 3 generations (each generation is 24 h), adding the schizochytrium strain into a culture medium with 2g/L propionate wastewater as the solvent according to an inoculum size ratio of 1%, domesticating for 30 generations, continuously adding the schizochytrium strain into a culture medium with 4g/L propionate wastewater as the solvent according to an inoculum size ratio of 1%, domesticating for 30 generations, continuously adding the schizochytrium strain into a culture medium with 6g/L propionate wastewater as the solvent according to an inoculum size ratio of 1%, domesticating for 30 generations, and continuously adding the schizochytrium strain into a culture medium with 8g/L propionate wastewater as the solvent according to an inoculum size ratio of 1%, domesticating for 30 generations. Meanwhile, 10ml of seed liquid after 2,4,6,8g/L propionate wastewater domestication is respectively taken for biomass measurement.
As a result, as shown in FIG. 3, domestication was successful, but eventually the propionic acid tolerance of schizochytrium reached only 6g/L, and after exceeding the concentration of 6g/L, schizochytrium was not substantially able to grow.
Example 3 comparison of lipid fractions obtained after fermentation of domesticated schizochytrium strains
Culturing fermentation seeds: after schizochytrium strains with high efficiency by utilizing propionate are inoculated in a flat-plate culture medium for culture, single bacterial colonies are selected and inoculated in a 250mL conical flask (containing 50mL of seed culture medium), and the first-stage seeds are obtained after shaking culture for 24 hours at the temperature of 28 ℃ and at the speed of 180 r/min. 1mL of the primary seed culture solution is inoculated into a 250mL conical flask (containing 50mL of seed culture medium), and shaking culture is carried out at 28 ℃ for 24h at 180r/min to obtain secondary seeds. 1mL of the secondary seed culture solution is inoculated into a 250mL conical flask (containing 50mL of seed culture medium), and shake cultivation is carried out for 24h at 28 ℃ and 180r/min to obtain tertiary seeds which are used as strains for fermentation. The seed liquid and the fermentation medium are respectively prepared by taking clear water and 6g/L propionate wastewater as solvents.
Shake flask fermentation culture: 10mL of the secondary seed culture solution is inoculated into a 500mL conical flask (containing 90mL of fermentation medium), shake cultivation is carried out for 48h at 28 ℃ and 180r/min, and the seed solution is taken for methyl esterification and carotenoid extraction.
The methyl esterification step is as follows:
(1) 20 mu L of fermentation medium lipid was taken and added to a 2mL EP tube;
(2) Adding the newly prepared 0.5MNaOH-CH3OH solution into an EP tube, and oscillating for 12 hours at room temperature at 1200 turns;
(3) The reaction was stopped by adding 50. Mu.L of concentrated sulfuric acid to the EP tube, and 1mL of n-hexane was added thereto and the mixture was shaken for 1 hour to extract the lipid;
(4) And taking normal hexane phase for gas phase detection.
The carotenoid extraction process is as follows:
(1) Taking the fermented culture medium, centrifuging to remove supernatant, precipitating at-80 ℃ for 12 hours, and freeze-drying by using a freeze dryer;
(2) Weighing 20mg of freeze-dried thalli, and grinding by a grinding machine;
(3) After extraction with 1ml of acetone, the liquid phase was detected.
The fatty acid composition results are shown in fig. 4, and the DHA content of the domesticated bacteria in the culture medium after the propionate is completely replaced is higher, and finally reaches 49.5%. In addition, as shown in FIG. 5, the total yield of carotenoids of the domesticated bacteria was increased by 1.91 times as compared with fermentation in a fermentation medium in which pure water was used as a solvent.
Example 4. Domesticated schizochytrium strains were subjected to fed-batch fermentation using propionic acid wastewater completely in place of the medium.
Except for the seed solution and the fermentation medium, the acid, alkali and sugar solution required by the feed fermentation are prepared by using propionate.
During the fed-batch fermentation, the propionate in the acid, base and sugar liquor will enter the fermentation system.
The domesticated schizochytrium limacinum is inoculated with a culture medium with the total substitution of 6g/L sodium propionate wastewater according to the proportion of 1 percent, and is cultivated for 24 hours at 28 ℃ and 170rpm, which is called first generation, and is continuously cultivated for 3 generations. Inoculating to fermentation liquid according to the proportion of 10%.
The fermenter was a Di BIL 5L fermenter. 3L of fermentation liquor with the total substitution of 6g/L propionic acid wastewater is prepared in a fermentation tank, sterilization is carried out for 30 minutes at 115 ℃, and seed liquor is inoculated after cooling is finished. The initial rotating speed is 300rpm, the ventilation amount is 2ppm, the temperature is kept at 28 ℃, and the dissolved oxygen is 15% -30%. The pH is controlled to be 6.5-7.0, the pH is regulated and controlled by using pre-prepared acid and alkali, the glucose concentration is sampled and measured every 12 hours, the glucose concentration in a fermentation tank is kept above 30g/L, and the fermentation tank is continuously cultured for 120 hours.
The acid, the alkali and the sugar used in the method are also prepared by using 6g/L propionic acid wastewater as a solvent, wherein the acid is citric acid monohydrate, and the alkali is sodium hydroxide;
the formula of the fermentation liquor is as follows: the pH value is 6.0-6.5, and comprises: 60-100g/L of glucose, 5-15 g/L of yeast extract powder, 5-12 g/L of sodium sulfate, 2-4 g/L of magnesium sulfate, 4-8 g/L of ammonium sulfate, 1-2 g/L of potassium chloride, 0.1-0.2 g/L of calcium chloride, 0.5-1 g/L of potassium sulfate, 0.5-2 g/L of monopotassium phosphate, 15-20 g/L of sodium glutamate, 1-5 mg/L of zinc sulfate heptahydrate, 0.01-0.1 mg/L of cobalt chloride hexahydrate, 2-6 mg/L of copper sulfate pentahydrate, 1-2 mg/L of nickel sulfate hexahydrate, 8-15 mg/L of ferric sulfate heptahydrate, 2-4 mg/L of calcium pantothenate, 3-5 mg/L of manganese chloride tetrahydrate, 0.04mg/L of sodium molybdate dihydrate, 63-10mg/L of vitamin B 12 0.1-0.8mg/L, the solvent is prepared by using 6g/L propionate waste water to completely replace clean water, sterilizing for 30 minutes at 115 ℃, and cooling.
As shown in FIG. 6, the result shows that the biomass of the domesticated bacteria in the propionic acid wastewater with the concentration of 6g/L reaches 106g/L, the grease content reaches 63g/L, and the DHA content is higher than 31g/L. Most importantly, when the domesticated bacteria ferment in the fully prepared culture medium of the propionic acid wastewater of 6g/L, the glucose consumption capability of schizochytrium is not affected, and finally, the domesticated bacteria consume 300 g/L of glucose within 120 h. As shown in Table 1, the accumulation of lipid and DHA production by the domesticated bacteria was superior to that of the homologous strain. Therefore, the strain obtained by utilizing the adaptive laboratory domestication means not only greatly improves the tolerance of propionic acid wastewater, but also can accumulate a large amount of DHA, thereby laying a powerful foundation for the subsequent fermentation of schizochytrium by directly utilizing industrial propionic acid wastewater.
Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments.

Claims (10)

1. Schizochytrium limacinum capable of efficiently utilizing propionate is characterized in that: the schizochytrium is obtained by taking propionate waste water as a base, preparing 2g/L, 4g/L, 6g/L and 8g/L propionate waste water by utilizing sodium propionate gradient, and preparing a complete substitution culture medium by using the propionate waste water as a solvent to carry out adaptive domestication on the schizochytrium.
2. Schizochytrium limacinum with efficient use of propionate according to claim 1, characterized in that: the domestication method comprises the following steps:
adding 1ml of schizochytrium strain frozen in a 20% glycerol pipe into a seed culture medium of normal schizochytrium seed solution, continuously culturing for 3 generations, culturing each generation for 24 hours, adding the schizochytrium strain into a seed culture medium with 2g/L propionate wastewater as a solvent according to an inoculum size ratio of 1%, domesticating for 30 generations, continuously adding the schizochytrium strain into a seed culture medium with 4g/L propionate wastewater as a solvent according to an inoculum size ratio of 1%, domesticating for 30 generations, continuously adding the schizochytrium strain into a seed culture medium with 6g/L propionate wastewater as a solvent according to an inoculum size ratio of 1%, domesticating for 30 generations, continuously adding the schizochytrium strain into a seed culture medium with 8g/L propionate wastewater as a solvent according to an inoculum size ratio of 1%, domesticating for 30 generations; simultaneously, respectively taking 2g/L, 4g/L, 6g/L and 8g/L of seed liquid after the domestication of the propionate wastewater is finished, and respectively carrying out biomass measurement;
After successful domestication, schizochytrium limacinum with propionic acid tolerance capacity reaching 6 g/L and efficient utilization of propionate is finally obtained.
3. Schizochytrium limacinum with efficient use of propionate according to claim 1, characterized in that: after the concentration of propionate exceeds 6 g/L, the schizochytrium cannot grow;
or the propionate wastewater is from bakery production wastewater, sodium propionate is used as a preservative in the bread, and the wastewater contains sodium propionate with the concentration of 2.4 g/L;
alternatively, the Schizochytrium strain frozen in 20% glycerol is Schizochytrium (Schizochytrium sp.) HX-308.
4. A schizochytrium limacinum with efficient propionate utilization according to any of claims 1 to 3, characterized in that: the seed culture medium of the normal schizochytrium limacinum seed solution is as follows: the pH of the medium was 6.4 and included: glucose 50g/L, yeast extract 5g/L, sodium sulfate 5g/L, magnesium sulfate 2 g/L, ammonium sulfate 6 g/L, potassium chloride 1 g/L, calcium chloride 0.1 g/L, potassium sulfate 0.6 g/L, potassium dihydrogen phosphate 1 g/L, sodium glutamate 10g/L, zinc sulfate heptahydrate 3 mg/L, cobalt chloride hexahydrate 0.05mg/L, copper sulfate pentahydrate 5mg/L, nickel sulfate hexahydrate 1 mg/L, iron sulfate heptahydrate 10mg/L, calcium pantothenate 4mg/L, manganese chloride tetrahydrate 5mg/L, sodium molybdate dihydrate 0.04mg/L; the solvent is prepared by using clear water;
The culture medium taking 2g/L propionate wastewater as a solvent is as follows: the pH of the medium was 6.4 and included: glucose 50g/L, yeast extract 5g/L, sodium sulfate 5g/L, magnesium sulfate 2g/L, ammonium sulfate 6 g/L, potassium chloride 1 g/L, calcium chloride 0.1 g/L, potassium sulfate 0.6 g/L, potassium dihydrogen phosphate 1 g/L, sodium glutamate 10g/L, zinc sulfate heptahydrate 3 mg/L, cobalt chloride hexahydrate 0.05mg/L, copper sulfate pentahydrate 5mg/L, nickel sulfate hexahydrate 1 mg/L, iron sulfate heptahydrate 10mg/L, calcium pantothenate 4mg/L, manganese chloride tetrahydrate 5mg/L, sodium molybdate dihydrate 0.04mg/L; the solvent is prepared by using 2g/L propionate wastewater to completely replace clear water;
the culture medium taking 4g/L propionate wastewater as a solvent is as follows: the pH of the medium was 6.4 and included: glucose 50g/L, yeast extract 5g/L, sodium sulfate 5g/L, magnesium sulfate 2g/L, ammonium sulfate 6 g/L, potassium chloride 1 g/L, calcium chloride 0.1 g/L, potassium sulfate 0.6 g/L, potassium dihydrogen phosphate 1 g/L, sodium glutamate 10g/L, zinc sulfate heptahydrate 3 mg/L, cobalt chloride hexahydrate 0.05mg/L, copper sulfate pentahydrate 5mg/L, nickel sulfate hexahydrate 1 mg/L, iron sulfate heptahydrate 10mg/L, calcium pantothenate 4mg/L, manganese chloride tetrahydrate 5mg/L, sodium molybdate dihydrate 0.04mg/L; the solvent is prepared by using 4g/L propionate wastewater to completely replace clear water;
The culture medium taking 6g/L propionate wastewater as a solvent is as follows: the pH of the medium was 6.4 and included: glucose 50g/L, yeast extract 5g/L, sodium sulfate 5g/L, magnesium sulfate 2 g/L, ammonium sulfate 6g/L, potassium chloride 1 g/L, calcium chloride 0.1 g/L, potassium sulfate 0.6 g/L, potassium dihydrogen phosphate 1 g/L, sodium glutamate 10g/L, zinc sulfate heptahydrate 3 mg/L, cobalt chloride hexahydrate 0.05mg/L, copper sulfate pentahydrate 5mg/L, nickel sulfate hexahydrate 1 mg/L, iron sulfate heptahydrate 10mg/L, calcium pantothenate 4mg/L, manganese chloride tetrahydrate 5mg/L, sodium molybdate dihydrate 0.04mg/L; the solvent is prepared by using 6g/L propionate wastewater to completely replace clear water;
the culture medium taking 8g/L propionate wastewater as a solvent is as follows: the pH of the medium was 6.4 and included: glucose 50g/L, yeast extract 5g/L, sodium sulfate 5g/L, magnesium sulfate 2 g/L, ammonium sulfate 6g/L, potassium chloride 1 g/L, calcium chloride 0.1 g/L, potassium sulfate 0.6 g/L, potassium dihydrogen phosphate 1 g/L, sodium glutamate 10g/L, zinc sulfate heptahydrate 3 mg/L, cobalt chloride hexahydrate 0.05mg/L, copper sulfate pentahydrate 5mg/L, nickel sulfate hexahydrate 1 mg/L, iron sulfate heptahydrate 10mg/L, calcium pantothenate 4mg/L, manganese chloride tetrahydrate 5mg/L, sodium molybdate dihydrate 0.04mg/L; the solvent is prepared by using 8g/L propionate wastewater to completely replace clear water.
5. Use of schizochytrium sp which utilizes propionate efficiently according to any of claims 1 to 4 for the treatment of propionate waste water and for the simultaneous production of DHA.
6. A method for treating propionate waste water and accumulating high-value DHA using schizochytrium efficiently utilizing propionate according to any of claims 1 to 4, characterized in that: the method comprises the following steps:
inoculating schizochytrium limacinum strains with high efficiency by utilizing propionate into a seed culture medium for activation to obtain strains for fermentation; inoculating a strain for fermentation into a fermentation culture medium for fermentation culture; collecting thallus to extract grease to obtain DHA.
7. The method according to claim 6, wherein: the whole period of the fermentation culture can reach 60 hours;
or the culture condition is 25-30 ℃ and 150-250 r/min shaking culture;
alternatively, the method for collecting the thalli and extracting the grease comprises the following steps:
1) Adding NaOH solution into fermentation broth after fermentation culture is finished to adjust pH=11-13, adding wall breaking enzyme with the mass final concentration of 0.01-0.4%, and oscillating for 5-15 h at 40-60 ℃ at 100-200 r/min;
2) Cooling to room temperature, and adding equal volume of absolute ethyl alcohol to inactivate wall-breaking enzyme;
3) Extracting with n-hexane, and collecting the upper organic phase;
4) Repeating the step 3) for several times, combining the organic phases, volatilizing the solvent to obtain the lipid.
8. The method according to claim 6, wherein: the seed culture medium and the fermentation culture medium are prepared by completely using propionate wastewater as a solvent;
alternatively, the strain for fermentation is obtained by the following method:
inoculating schizochytrium limacinum strain with high efficiency by using propionate into a flat-plate culture medium for culture, picking single bacterial colony, inoculating into a seed culture medium for activation, and culturing to obtain first-stage seeds; inoculating the first-level seeds into a seed culture medium, and culturing to obtain second-level seeds; inoculating the secondary seeds into a seed culture medium, and culturing to obtain tertiary seeds serving as strains for fermentation.
9. The method according to claim 8, wherein: the plate culture medium is as follows: the pH value of the culture medium is 6.0-6.5, and the culture medium comprises: 15-20g/L of agar, 30-60g/L of glucose, 8-15 g/L of yeast extract powder and sodium sulfate10-15 g/L of magnesium sulfate 2-4 g/L, ammonium sulfate 6-12 g/L, potassium chloride 1-2 g/L, calcium chloride 0.1-0.2 g/L, potassium sulfate 0.5-1 g/L, monopotassium phosphate 0.5-2 g/L, sodium glutamate 8-12 g/L, zinc sulfate heptahydrate 1-5 mg/L, cobalt chloride hexahydrate 0.01-0.1 mg/L, copper sulfate pentahydrate 2-6 mg/L, nickel sulfate hexahydrate 1-2 mg/L, ferric sulfate heptahydrate 8-15 mg/L, calcium pantothenate 2-4 mg/L, manganese chloride tetrahydrate 3-5 mg/L, sodium molybdate dihydrate 0.04mg/L, vitamin B 6 3-10 mg/L, vitamin B 12 0.3-1.2 mg/L; the solvent is prepared by using 6g/L propionate wastewater to completely replace clear water;
the seed culture medium is as follows: the pH value of the culture medium is 6.0-6.5, and the culture medium comprises: 40-60g/L of glucose, 4-6 g/L of yeast extract powder, 5-8 g/L of sodium sulfate, 2-4 g/L of magnesium sulfate, 4-8 g/L of ammonium sulfate, 1-2 g/L of potassium chloride, 0.1-0.2 g/L of calcium chloride, 0.5-1 g/L of potassium sulfate, 0.5-2 g/L of monopotassium phosphate, 8-12 g/L of sodium glutamate, 1-5 mg/L of zinc sulfate heptahydrate, 0.01-0.1 mg/L of cobalt chloride hexahydrate, 2-6 mg/L of copper sulfate pentahydrate, 1-2 mg/L of nickel sulfate hexahydrate, 8-15 mg/L of ferric sulfate heptahydrate, 2-4 mg/L of calcium pantothenate, 3-5 mg/L of manganese chloride tetrahydrate and 0.04mg/L of sodium molybdate dihydrate; the solvent is prepared by using 6g/L propionate wastewater to completely replace clear water;
the fermentation medium is as follows: the pH value of the culture medium is 6.0-6.5, and the culture medium comprises: 60-100g/L glucose, 5-15 g/L yeast extract powder, 5-12 g/L sodium sulfate, 2-4 g/L magnesium sulfate, 4-8 g/L ammonium sulfate, 1-2 g/L potassium chloride, 0.1-0.2 g/L calcium chloride, 0.5-1 g/L potassium sulfate, 0.5-2 g/L potassium dihydrogen phosphate, 15-20 g/L sodium glutamate, 1-5 mg/L zinc sulfate heptahydrate, 0.01-0.1 mg/L cobalt chloride hexahydrate, 2-6 mg/L copper sulfate pentahydrate, 1-2 mg/L nickel sulfate hexahydrate, 8-15 mg/L ferric sulfate heptahydrate, 2-4 mg/L calcium pantothenate, 3-5 mg/L manganese chloride tetrahydrate, 0.04mg/L sodium molybdate dihydrate, and vitamin B 6 3-10 mg/L, vitamin B 12 0.1-0.8mg/L; the solvent is prepared by using 6g/L propionate wastewater to completely replace clean water.
10. A method for accumulating high value DHA and biomass by fermentation of schizochytrium using schizochytrium efficiently utilizing propionate according to any of claims 1 to 4 and replacing the medium with entirely propionate waste water, characterized in that: the method comprises the following steps:
inoculating schizochytrium limacinum which efficiently utilizes propionate into a seed culture medium with 6g/L sodium propionate wastewater completely replaced according to a proportion of 1%, culturing at 28 ℃ and 170rpm for 24 hours, namely, continuously culturing for 3 generations; inoculating 10% of the mixed solution into fermentation liquid for fermentation, wherein the initial rotation speed is 300rpm, the ventilation amount is 2ppm, the temperature is kept at 28 ℃, and the dissolved oxygen is 15% -30%; controlling the pH value to be 6.5-7.0, performing pH regulation by using pre-prepared acid and alkali, sampling and measuring the glucose concentration every 12 hours, keeping the glucose concentration in a fermentation tank to be above 30g/L, and continuously culturing for 120 hours;
the acid, the alkali and the sugar used in the method are also prepared by using 6g/L propionic acid wastewater as a solvent, wherein the acid is citric acid monohydrate, and the alkali is sodium hydroxide;
the formula of the fermentation liquor is as follows: the pH value is 6.0-6.5, and comprises: 60-100g/L glucose, 5-15 g/L yeast extract powder, 5-12 g/L sodium sulfate, 2-4 g/L magnesium sulfate, 4-8 g/L ammonium sulfate, 1-2 g/L potassium chloride, 0.1-0.2 g/L calcium chloride, 0.5-1 g/L potassium sulfate, 0.5-2 g/L potassium dihydrogen phosphate, 15-20 g/L sodium glutamate, 1-5 mg/L zinc sulfate heptahydrate, 0.01-0.1 mg/L cobalt chloride hexahydrate, 2-6 mg/L copper sulfate pentahydrate, 1-2 mg/L nickel sulfate hexahydrate, 8-15 mg/L ferric sulfate heptahydrate, 2-4 mg/L calcium pantothenate, 3-5 mg/L manganese chloride tetrahydrate, 0.04mg/L sodium molybdate dihydrate, 6 3-10 mg/L vitamin B6 3-10 12 0.1-0.8mg/L, the solvent is prepared by using 6g/L propionate waste water to completely replace clean water, sterilizing for 30 minutes at 115 ℃, and cooling.
CN202310313325.4A 2023-03-28 2023-03-28 Schizochytrium limacinum with efficient utilization of propionate, application and method for treating propionate wastewater and accumulating high-value DHA Pending CN116445293A (en)

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