CN113373056A - Method for producing fucoxanthin by coupling marine microalgae culture and culture wastewater purification - Google Patents

Method for producing fucoxanthin by coupling marine microalgae culture and culture wastewater purification Download PDF

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CN113373056A
CN113373056A CN202110443711.6A CN202110443711A CN113373056A CN 113373056 A CN113373056 A CN 113373056A CN 202110443711 A CN202110443711 A CN 202110443711A CN 113373056 A CN113373056 A CN 113373056A
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culture
fucoxanthin
phaeodactylum tricornutum
livestock
aquaculture wastewater
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黄建科
江金顺
范江鹏
张超
余昕玥
王灿
秦萌
廖静
徐雪峰
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Hohai University HHU
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/32—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae
    • C02F3/322—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae use of algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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    • C02F2101/30—Organic compounds
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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    • C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/20—Nature of the water, waste water, sewage or sludge to be treated from animal husbandry
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    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids

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Abstract

The invention relates to a method for coupling livestock and poultry breeding wastewater treatment and phaeodactylum tricornutum culture, which comprises the following steps: (1) culturing Phaeodactylum tricornutum algae seeds in a microalgae mixotrophic culture mode; (2) inoculating phaeodactylum tricornutum algae liquid into livestock and poultry breeding wastewater to perform microalgae photoautotrophic culture; (3) after the culture is finished, collecting and obtaining phaeodactylum tricornutum, and recycling or discharging the cultured clear liquid; (4) fucoxanthin in the phaeodactylum tricornutum algae cells is obtained by a separation and extraction method. According to the method, the COD, ammonia nitrogen, total nitrogen and total phosphorus concentration of the wastewater are obviously reduced, and the removal of the COD, the nitrogen removal and the phosphorus removal of the wastewater are realized. Meanwhile, the concentration of algal cells and the concentration of fucoxanthin in the culture solution are obviously increased, the wastewater resources are biologically converted into the fucoxanthin with high added value, and the waste is changed into valuable.

Description

Method for producing fucoxanthin by coupling marine microalgae culture and culture wastewater purification
Technical Field
The invention relates to the field of culture wastewater treatment and algae culture, in particular to a method for producing fucoxanthin by coupling marine microalgae culture and culture wastewater purification.
Background
The culture wastewater refers to a general term of urine produced by a farm, whole excrement or residual excrement and feed residue, flushing water and wastewater produced during life and production of workers, wherein the flushing water accounts for the majority. The treatment difficulty of the livestock and poultry wastewater is high, and the method has the following characteristics: COD, SS, NH3high-N content; the biodegradability is good, and the precipitation performance is good; the water quality and the water quantity change greatly; it contains pathogenic bacteria and has offensive odor. At present, livestock and poultry breeding in China develops rapidly, about 45 hundred million tons of livestock and poultry manure are produced every year, and the Chemical Oxygen Demand (COD) of the livestock and poultry manure exceeds the sum of industrial wastewater and domestic sewage in China. Therefore, livestock breeding pollution is the third pollution source after industrial pollution and living pollution. The main treatment methods of the aquaculture wastewater include physical and chemical treatment technologies such as adsorption, magnetic flocculation precipitation, electrochemical oxidation and the like, and traditional biochemical methods such as microorganism anaerobic-aerobic treatment and the like, but the methods have the defects of complex treatment process, high treatment cost and resourceLow recycling rate and the like.
The microalgae is an original microorganism, has the advantages of strong environmental adaptability, high growth rate and the like, is rich in various nutritional ingredients such as protein, carotenoid, unsaturated fatty acid, vitamin, mineral elements and the like, and is widely applied to various fields such as biological medicine, nutritional functional food, food and food additive, feed and animal health care, aquaculture and the like. Meanwhile, the microalgae can rapidly utilize nutrient substances such as organic matters, nitrogen, phosphorus and the like to grow and reproduce, and the microalgae can remove COD (chemical oxygen demand) and BOD (biochemical oxygen demand) in the wastewater and realize nitrogen and phosphorus removal. A microalgae-based wastewater treatment technology is a hotspot of current domestic and foreign researches, and the technology can realize the purification of wastewater and synchronously obtain high-added-value microalgae biomass which can be directly used as feed, bait and fertilizer; or separating and extracting natural active substances in microalgae, such as fucoxanthin, xanthophyll, astaxanthin, polysaccharide and unsaturated fatty acid, to obtain high value-added product.
Fucoxanthin is a carotenoid widely distributed in marine microalgae, such as Phaeodactylum tricornutum, and Chlorella rhombifolia. Research shows that fucoxanthin has a plurality of remarkable efficacies, such as antioxidation, antitumor action, anticancer action, cerebrovascular protection action, osteoporosis prevention action and the like. Therefore, the fucoxanthin has wide application value, and crop functional nutritional supplements and anti-obesity agents are available at present.
Accordingly, aiming at the problems of difficult wastewater treatment and low resource utilization in the breeding industry, the existing breeding wastewater treatment method needs to be improved, and the prior art does not disclose a method or technology for purifying the breeding wastewater and producing fucoxanthin by adopting phaeodactylum tricornutum.
Disclosure of Invention
In order to overcome the defects, the invention aims to provide a method for producing fucoxanthin by coupling marine microalgae culture and culture wastewater purification in the field, so that the technical problems of complex treatment process, high cost, low resource recovery and utilization rate and low additional output value of the existing culture wastewater are solved. The purpose is realized by the following technical scheme.
A method for producing fucoxanthin by coupling marine microalgae culture and culture wastewater purification is characterized by comprising the following steps:
(1) culturing the marine microalgae phaeodactylum tricornutum algae species in a microalgae mixotrophic culture mode;
(2) inoculating phaeodactylum tricornutum algae liquid into livestock and poultry breeding wastewater to perform microalgae photoautotrophic culture;
(3) after the culture is finished, collecting and obtaining phaeodactylum tricornutum, and recycling or discharging the cultured clear liquid;
(4) fucoxanthin in the phaeodactylum tricornutum algae cells is obtained by a separation and extraction method.
In the step (1), the mixotrophic culture mode adopts glycerol as a carbon source, peptone as a nitrogen source, the concentration of the carbon source is 0.1-0.5 mmol/L, the concentration of the peptone is 0.5g/L, and 1g/L of chlorella hot water extract is added.
In the step (2), the livestock and poultry breeding wastewater is diluted to a COD concentration of 400-1700 mg/L, an ammonia nitrogen concentration of 90-300 mg/L and a total phosphorus concentration of 2-5 mg/L, the dilution method is to adopt seawater with a salinity of 25 thousandths or adopt supernatant recycled in the third step, and the dilution multiple of the livestock and poultry breeding wastewater is 3-12 times.
The temperature for culturing the microalgae is 20-25 ℃, and the culture period is 14-16 days.
And adjusting the pH value of the livestock and poultry breeding wastewater to 6.5-7.5.
The inoculation amount of the phaeodactylum tricornutum algae seed solution is 25% of the volume of the livestock and poultry breeding wastewater.
Before the step (2), the livestock and poultry breeding wastewater is subjected to physical sedimentation and membrane filtration to remove particle suspended matters and impurities, and is subjected to disinfection and sterilization treatment, wherein the disinfection and sterilization are carried out by any one or more combined modes of a sodium hypochlorite method, an ultraviolet disinfection method and a microfiltration membrane filtration method.
The phaeodactylum tricornutum is cultured under the condition of keeping out of the sun or illumination, and when the phaeodactylum tricornutum is cultured under the illumination condition, the illumination intensity is 1000-5000 lux; the device adopted by the culture comprises a shake flask, a fermentation tank or a sterilizable photobioreactor, and when the culture device is the shake flask, the rotating speed of a shaking table is controlled to be 100-200 rpm; when the culture device is a fermentation tank or a sterilizable photobioreactor, stirring and ventilation are started, and the dissolved oxygen is controlled to be not less than 10%.
In the step (3), microalgae is obtained by any one of a centrifugal method, an air floatation method and a flocculation method.
In the step (4), ethanol extraction or supercritical CO extraction is carried out2The extraction method is used for extracting fucoxanthin from Phaeodactylum tricornutum species.
The invention uses the Phaeodactylum tricornutum to rapidly utilize the organic matters, nitrogen, phosphorus and other nutrient substances in the aquaculture wastewater for growth and reproduction, thereby achieving the effects of removing COD and BOD in the aquaculture wastewater and removing nitrogen and phosphorus, realizing high-efficiency purification treatment of the wastewater, and simultaneously extracting the high-added-value microalgae biomass fucoxanthin by the Phaeodactylum tricornutum, wherein the fucoxanthin has a plurality of remarkable effects, such as antioxidation, anti-tumor effect, anti-cancer effect, cerebrovascular protection effect, osteoporosis prevention effect and the like, so the application of the fucoxanthin is quite wide, and the application value is higher. In addition, the phaeodactylum tricornutum culture solution in the wastewater treatment can be recycled, so that the low-cost high-culture wastewater treatment efficiency is realized, and most of the culture wastewater is highly recycled. It is suitable for being used as the existing livestock breeding wastewater treatment method or the improvement of the similar wastewater treatment method.
Drawings
FIG. 1 is a schematic flow diagram of the process of the present invention.
FIG. 2 is a graph showing the change of the cell dry weight, COD, ammonia nitrogen and total phosphorus concentration of the algae cultured by Phaeodactylum tricornutum of example 1 of the present invention.
FIG. 3 is a graph showing the change of fucoxanthin concentration in Phaeodactylum tricornutum culture according to example 1 of the present invention.
FIG. 4 is a graph showing the change of the cell dry weight, COD, ammonia nitrogen and total phosphorus concentration of the algae cultured by Phaeodactylum tricornutum of example 2.
FIG. 5 is a graph showing the change of fucoxanthin concentration in Phaeodactylum tricornutum culture in example 2 of the present invention.
Detailed Description
The invention will now be further described with reference to the accompanying drawings.
Example 1: as shown in FIG. 1, which is a flow chart of the method of the present invention, firstly, the Phaeodactylum tricornutum algae species are cultured in the microalgae mixotrophic culture mode, wherein glycerol is used as a carbon source, peptone is used as a nitrogen source, the concentration of the carbon source is 0.1-0.5 mmol/L, the concentration of the peptone is 0.5g/L, and 1g/L of hot water extract of chlorella is added. Then standing and settling the livestock and poultry breeding wastewater for 3-5 hours, filtering with a microfiltration membrane, diluting by 3 times with seawater with salinity of 25 thousandth to ensure that the COD concentration in the livestock and poultry breeding wastewater is 1700mg/L, ammonia nitrogen is 300mg/L, total phosphorus is 5mg/L, and the pH is adjusted to 6.5-7.5. 200mL of the liquid was put into a 500 mL-sized shake flask, and then sterilized at 115 ℃ for 25min at high temperature. After cooling, 25 mL of phaeodactylum tricornutum algae seed liquid is inoculated, the initial inoculation density is 0.27g/L, then the shake flask is placed in a shaking table, the rotation speed is adjusted to 150rpm, and the culture temperature is controlled to be 25 ℃.
In the process, the change process of the algae cell concentration, COD, ammonia nitrogen and total phosphorus under the dry weight condition is measured. As can be seen from FIG. 2, the algal cells rapidly grow after inoculation, and the concentration of the Phaeodactylum tricornutum algae reaches 0.9g/L after the culture to the 16 th day; COD of the wastewater is reduced from 1700mg/L to 812mg/L, and ammonia nitrogen and total phosphorus are respectively reduced from 300mg/L and 5mg/L to 130mg/L and 1.3 mg/L. Meanwhile, as can be seen from FIG. 3, the fucoxanthin concentration of the Phaeodactylum tricornutum culture solution rapidly increased from 2.7mg/L to 13.5 mg/L. Therefore, the phaeodactylum tricornutum can quickly remove COD, ammonia nitrogen and total phosphorus in the wastewater, realize the purification of the wastewater and simultaneously can produce a natural product fucoxanthin with high added value.
Example 2: firstly, adopting a microalgae mixotrophic culture mode to culture Phaeodactylum tricornutum algae seeds, adopting glycerol as a carbon source and peptone as a nitrogen source in the mixotrophic culture mode, wherein the concentration of the carbon source is 0.1-0.5 mmol/L, the concentration of the peptone is 0.5g/L, and adding 1g/L of chlorella hot water extract. Then the livestock and poultry breeding wastewater is statically settled for 3-5 hours, and then is filtered by a microfiltration membrane, sterilized by ultraviolet light, and diluted by 12 times by seawater with salinity of 25 thousandth, so that the COD concentration in the livestock and poultry breeding wastewater is 400mg/L, the ammonia nitrogen is 89mg/L, and the total phosphorus is 2.0 mg/L. Then, 20 mL of the Phaeodactylum tricornutum algae strain was inoculated into a 500mL triangular shake flask (liquid loading 200 mL) at an initial inoculation density of 0.27g/L, and the shake flask was placed in a shaker at a rotation speed of 150rpm with the culture temperature controlled at 25 ℃.
In the process, the change process of the algae cell concentration, COD, ammonia nitrogen and total phosphorus under the dry weight condition is measured. As can be seen from FIG. 2, the algal cells rapidly grow after inoculation, and the concentration of the Phaeodactylum tricornutum algae reaches 1.22g/L after the culture to the 16 th day; COD of the wastewater is reduced from 400mg/L to 200mg/L, and ammonia nitrogen and total phosphorus are reduced from 89mg/L and 2mg/L to 0.1mg/L and 0.52mg/L respectively. Meanwhile, as can be seen from FIG. 5, the fucoxanthin concentration of the Phaeodactylum tricornutum culture solution rapidly increased from 2.7mg/L to 24.4 mg/L. Therefore, the phaeodactylum tricornutum can quickly remove COD, ammonia nitrogen and total phosphorus in the wastewater, realize the purification of the wastewater and simultaneously can produce a natural product fucoxanthin with high added value.
The above description is intended to illustrate the technical means of the present invention, and not to limit the technical scope of the present invention. Modifications of the invention which are obvious to those skilled in the art in view of the prior art are also within the scope of the invention as claimed.

Claims (10)

1. A method for producing fucoxanthin by coupling marine microalgae culture and culture wastewater purification is characterized by comprising the following steps:
(1) culturing the marine microalgae phaeodactylum tricornutum algae species in a microalgae mixotrophic culture mode;
(2) inoculating phaeodactylum tricornutum algae liquid into livestock and poultry breeding wastewater to perform microalgae photoautotrophic culture;
(3) after the culture is finished, collecting and obtaining phaeodactylum tricornutum, and recycling or discharging the cultured clear liquid;
(4) fucoxanthin in the phaeodactylum tricornutum algae cells is obtained by a separation and extraction method.
2. The method for producing fucoxanthin by coupling marine microalgae culture and culture wastewater purification according to claim 1, wherein in the step (1), glycerol is used as a carbon source, peptone is used as a nitrogen source, the concentration of the carbon source is 0.1-0.5 mmol/L, the concentration of the peptone is 0.5g/L, and 1g/L of chlorella hot water extract is added.
3. The method for producing fucoxanthin by coupling marine microalgae culture and aquaculture wastewater purification according to claim 1, wherein in the step (2), the livestock and poultry aquaculture wastewater is diluted to a COD concentration of 400-1700 mg/L, an ammonia nitrogen concentration of 90-300 mg/L and a total phosphorus concentration of 2-5 mg/L, the dilution method is to adopt seawater with a salinity of 25 thousandth or a supernatant recycled in the third step, and the dilution multiple of the livestock and poultry aquaculture wastewater is 3-12 times.
4. The method for producing fucoxanthin by coupling marine microalgae culture and aquaculture wastewater purification according to claim 1, wherein the temperature of microalgae culture is 20-25 ℃ and the culture period is 14-16 days.
5. The method for producing fucoxanthin by coupling marine microalgae culture and aquaculture wastewater purification according to claim 1, wherein the pH of the livestock and poultry aquaculture wastewater is adjusted to 6.5-7.5.
6. The method for producing fucoxanthin by coupling marine microalgae cultivation with aquaculture wastewater purification according to claim 1, wherein the amount of the Phaeodactylum tricornutum strain liquid is 25% of the volume of the livestock and poultry aquaculture wastewater.
7. The method for producing fucoxanthin by coupling marine microalgae culture and aquaculture wastewater purification according to claim 1, wherein before the step (2), the livestock and poultry aquaculture wastewater is subjected to physical sedimentation and membrane filtration to remove particle suspended matters and impurities, and is subjected to disinfection and sterilization treatment by any one or a combination of two or more of sodium hypochlorite method, ultraviolet disinfection method and microfiltration membrane filtration method.
8. The method for producing fucoxanthin by coupling marine microalgae culture and aquaculture wastewater purification according to claim 1, wherein the phaeodactylum tricornutum is cultured under a dark or light condition, and when the phaeodactylum tricornutum is cultured under the light condition, the light intensity is 1000-5000 lux; the device adopted by the culture comprises a shake flask, a fermentation tank or a sterilizable photobioreactor, and when the culture device is the shake flask, the rotating speed of a shaking table is controlled to be 100-200 rpm; when the culture device is a fermentation tank or a sterilizable photobioreactor, stirring and ventilation are started, and the dissolved oxygen is controlled to be not less than 10%.
9. The method for producing fucoxanthin by coupling marine microalgae culture and aquaculture wastewater purification according to claim 1, wherein in the step (3), microalgae is obtained by any one of centrifugation, air flotation and flocculation.
10. The method for producing fucoxanthin in combination of marine microalgae culture and aquaculture wastewater purification as claimed in claim 1, wherein in said step (4), ethanol extraction or supercritical CO extraction is used2The extraction method is used for extracting fucoxanthin from Phaeodactylum tricornutum species.
CN202110443711.6A 2021-04-23 2021-04-23 Method for producing fucoxanthin by coupling marine microalgae culture and culture wastewater purification Pending CN113373056A (en)

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