CN114538619A - Method for purifying mariculture tail water by using heterotrophic microalgae - Google Patents
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Abstract
The invention relates to a method for purifying seawater culture tail water by utilizing heterotrophic microalgae, which belongs to the field of seawater culture tail water treatment, wherein the microalgae is chlorella pyrenoidosa, the chlorella cultured by heterotrophic fermentation is centrifuged and washed for 2 times, photoautotrophic conversion and salinity acclimation are carried out, the microalgae after photoautotrophic conversion and salinity acclimation are inoculated into the culture tail water for culture, and the inoculation density is 1 multiplied by 105‑106So that the microalgae cells can purify the culture discharged wastewater in a synergic way in the culture process(ii) a The heterotrophic chlorella pyrenoidosa is subjected to light conversion and salinity conversion by adopting heterotrophic fermentation, so that the heterotrophic chlorella pyrenoidosa has the capability of treating inorganic carbon, nitrogen, phosphorus and other nutrient salts, can be applied to seawater culture tail water purification, and avoids the defects of low photoautotrophic culture density, unstable culture condition and large collection difficulty of microalgae.
Description
Technical Field
The invention relates to a method for purifying tail water of mariculture by using heterotrophic microalgae, which belongs to the field of tail water treatment of mariculture and can be applied to tail water treatment production of seawater prawn culture ponds and industrial fish culture.
Background
In 2020, the total output of aquaculture in China reaches 5224 ten thousand tons, which accounts for 80% of the total output of aquatic products, and with the increase of economic development and the demand of aquatic products, aquaculture in China tends to the development of intensive and large-scale aquaculture modes with high density and high input, and new challenges are brought to standard discharge of aquaculture tail water. Research has shown that in mariculture, 52-95% of nitrogen, 85% of phosphorus and 80-88% of carbon in the feed are lost into a culture water body in the modes of feed waste, biological excretion, respiration and the like to generate culture tail water with high-concentration pollutants, wherein the main pollutants comprise ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, inorganic phosphorus, organic matters and fouling organisms. The inorganic nitrogen and phosphorus are main stress factors influencing aquaculture environment and organisms, so that on one hand, the inorganic nitrogen and phosphorus can stress cultured animals to cause stress reaction to reduce disease resistance, and on the other hand, the inorganic nitrogen and phosphorus can cause eutrophication of water quality of surrounding water areas to cause disasters such as water bloom, red tide and the like, and the healthy and sustained development of aquaculture industry is restricted. How to effectively utilize and treat the breeding tail water resources and implement the standard discharge of the breeding tail water is a key problem which needs to be solved urgently to realize the green development of the breeding industry in China.
At present, the traditional culture tail water treatment method comprises a physical treatment method, a chemical treatment method, a biomembrane method, an electrochemical method, an artificial wetland treatment method and the like. The methods generally have the problems of high construction cost, high facility operating cost and the like, so that the popularization and the application of the methods are influenced. In recent years, a large number of studies on microalgae culture and wastewater treatment have been conducted at home and abroad, and algae treatment technologies have been developed. Microalgae is a general term for a class of microorganisms that contain chlorophyll a and can perform photosynthesis, and can grow by using carbon, nitrogen and phosphorus in water and synthesize cell components such as proteins and nucleic acids required by the microalgae. In the aquaculture industry, microalgae is widely applied to various fields of aquatic organism baits, feed additives and the like as primary productivity, and plays roles in promoting nutrient substance circulation, reducing feed coefficient, improving survival rate and the like. Therefore, the microalgae is utilized for the treatment of the culture tail water, on one hand, the water quality can be purified, the nitrogen and phosphorus nutrient salts in the culture tail water can be effectively removed, on the other hand, the microalgae can be used as primary productivity to provide biological bait, and the microalgae has important application and popularization values.
The large-scale autotrophic culture of microalgae is mainly carried out by adopting a photobioreactor, a pond open culture mode and the like, depends on algal cell chloroplasts and utilizes solar energy to convert CO into CO2And water is used for forming organic substances to perform algae cell amplification, and nutritive salt is an inorganic carbon source, so that the culture mode has the defects of large occupied area, long production period, low cell density, uncontrollable culture conditions, easiness in pollution, unstable quality and the like, the subsequent collection and utilization cost is high, and the application of the autotrophic microalgae in the treatment of aquaculture tail water is limited.
The Chlorella pyrenoidosa can utilize light energy and CO2In addition to normal photoautotrophic growth, organic carbon source and O can be used under no light condition2Carrying out heterotrophic growth and propagation. The heterotrophic culture algae cell proliferation rate is faster than that of photoautotrophic conditions, the culture conditions do not need an illumination auxiliary system and are not influenced by environmental factors, the culture yield of unit water body can be greatly improved, and the cost for harvesting and utilizing algae cells is far lower than that of autotrophic culture due to extremely high density of algae cells, so that the cost can be reduced by utilizing heterotrophic microalgae to carry out culture tail water treatment. However, heterotrophic microalgae cannot be directly applied to culture tail water treatment and does not have the capability of utilizing nutrient salts such as inorganic carbon, nitrogen, phosphorus and the like.
At present, no report on a method for purifying the tail water of mariculture by using heterotrophic microalgae is found. The authorization notice number CN101531978B discloses a method for regulating and controlling microalgae mixed culture and synergistically purifying culture discharge water by using lactobacillus, wherein the microalgae used are photoautotrophic chlorella, cryptophyceae and green oscillatoria. Application publication No. CN110627213A discloses a method for efficiently treating high ammonia nitrogen wastewater by a microalgae photo-fermentation method, which comprises the step of placing the high ammonia nitrogen wastewater after blending into a photo-fermentation tank for removing ammonia nitrogen.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for purifying mariculture tail water by using heterotrophic microalgae, which is characterized in that the inoculation density of the microalgae is adjusted by photoautotrophic conversion of the heterotrophic microalgae, trace elements are added, the rapid growth of the microalgae in the mariculture tail water is promoted, and the purpose of removing nitrogen and phosphorus nutritive salts from the mariculture tail water is achieved.
The invention is realized by the following technical scheme;
a method for purifying seawater culture tail water by heterotrophic microalgae comprises centrifuging and washing chlorella cultured by heterotrophic fermentation for 2 times, performing photoautotrophic conversion and salinity acclimation, inoculating the microalgae after photoautotrophic conversion and salinity acclimation into culture tail water, and culturing at an inoculation density of 1 × 105-106The microalgae cells are cooperatively purified to culture the discharged wastewater in the culture process;
the photoautotrophic conversion method comprises the following steps: diluting the washed microalgae by 1000 times according to the volume ratio, adding a complete BG11 culture medium, wherein the culture conditions are that the temperature is 25-28 ℃, the illumination intensity is 5000-;
the salinity domestication method comprises the following steps: the salinity of the heterotrophic fermentation medium is 10 per mill, and NaCl with corresponding weight is added to the required final concentration according to the salinity increased by 5 per mill every day. The desired final concentration is the salinity of the wastewater to be treated using microalgae.
Further, the final concentration of the salinity condition is consistent with the concentration of the wastewater to be treated.
Further, photoautotrophic conversion simultaneously carries out salinity acclimation of heterotrophic microalgae.
Further, the heterotrophic fermentation culture method of the chlorella pyrenoidosa comprises the following steps: inoculating chlorella pyrenoidosa algae liquid which is subjected to aseptic culture in logarithmic growth phase into a bioreactor, wherein a fermentation culture medium comprises 30g/L of glucose, 25g/L of yeast extract, 0.5g/L of potassium dihydrogen phosphate, 0.5g/L of magnesium sulfate, 0.5g/L of sodium citrate, 130 mg/L of vitamin B, 630 mg/L of vitamin B, 125 mg/L of vitamin B and 2mg/L of biotin, the pH value is 6.5, the temperature is 30 ℃, and the continuous fermentation culture is carried out for 120 hours.
Further, in the process of cooperatively purifying culture wastewater discharged by microalgae culture and aquaculture, other nutrient salts except nitrogen and phosphorus are added according to a BG11 culture medium to ensure the growth and metabolism of microalgae, and the addition amount of each nutrient salt is MgSO4·7H20-375mg/L、CaCl2·2H2O-180mg/L、Na2CO3180mg/L, 30mg/L of citric acid, 30mg/L of ferric citrate and 1mL/L of trace element A5 solution.
Compared with the prior art, the invention has the beneficial effects that:
the heterotrophic chlorella pyrenoidosa is subjected to photoconversion and salinity conversion, so that the heterotrophic chlorella pyrenoidosa has the capability of treating inorganic carbon, nitrogen, phosphorus and other nutrient salts, can be applied to seawater culture tail water purification, avoids the defects of low photoautotrophic culture density, unstable culture condition and high harvesting difficulty of microalgae, can form a standardized treatment flow for heterotrophic fermented microalgae, can realize nitrogen removal and phosphorus removal of culture tail water in situ through photoautotrophic conversion and salinity acclimation, realizes accumulation and acquisition of primary productivity, realizes continuous culture of prawns, saves water resources, is economical and practical, and is suitable for wide popularization. The photoautotrophic growth characteristic of the chlorella pyrenoidosa is fully considered, and other nutritive salts except nitrogen and phosphorus are added into the culture water body, so that the nutritional requirements of growth and proliferation of algae cells are met, the proliferation rate is ensured, and the efficiency of removing nitrogen, phosphorus and purifying water quality is improved.
The method utilizes an industrial fermentation method to perform heterotrophic culture on the microalgae, takes organic matters as a carbon source and a nitrogen source to perform heterotrophic culture, is easy to control the culture conditions, effectively avoids the pollution of the mixed algae, bacteria and protozoa, and can greatly save the space, improve the culture yield and save the harvesting cost.
Detailed Description
The following examples are merely illustrative of the present invention, and the scope of the present invention is not limited in any way by the examples. The objectives of the present invention can be achieved by the ordinary skilled person in the art according to the disclosure of the present invention and the ranges of the parameters.
Example 1
Heterotrophic fermentation culture of chlorella pyrenoidosa: inoculating 50mL of sterile chlorella pyrenoidosa solution into a 500mL shake flask filled with 100mL of culture medium, and culturing for 48h in a shaking table at 30 ℃ at the rotating speed of 200rpm to obtain chlorella pyrenoidosa in logarithmic growth phase; adding the algae solution into a bioreactor, adding 1.2L fermentation medium into a 3L bioreactor, wherein the fermentation medium is glucose 30g/L, yeast extract 25g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, sodium citrate 0.5g/L, vitamin B130mg/L, vitamin B630mg/L, vitamin B125mg/L and biotin 2mg/L, controlling the temperature at 30 ℃ in the fermentation process in the fermentation medium of the bioreactor, automatically adding 2M NaOH to keep the pH value at 6.5, and carrying out co-fermentation culture for 120 hours.
Collecting Chlorella pyrenoidosa cultured for 96h, centrifuging at rotation speed of 5000-6000rpm for 8-10min, discarding supernatant, washing with sterile 10 ‰ saline water, repeating for 2 times, suspending with sterile 10 ℃saline water, and allowing cell density of Chlorella pyrenoidosa to be greater than 1012cell/mL。
Diluting the washed microalgae by 1000 times according to the volume ratio, adding a full-value BG11 culture medium for photoautotrophic conversion, wherein the addition amount of nutrient salts in the BG11 culture medium is NaNO3-1500mg/L、KH2PO4-400mg/L、MgSO4·7H20-375mg/L、CaCl2·2H2O-180mg/L、Na2CO3180mg/L, 30mg/L of citric acid, 30mg/L of ferric citrate and 5-1mL/L of trace element solution A.
The photoautotrophic conversion culture conditions comprise the temperature of 25-28 ℃, the illumination intensity of 5000-.
The photoautotrophic conversion simultaneously carries out the salinity domestication of the heterotrophic microalgae, and the domestication method comprises the following steps: NaCl (analytically pure, Chinese medicine) with the corresponding weight is added according to the salinity increased by 5 per day, and the final salinity concentration of the embodiment is 26 per mill, namely, after 5 days of salinity acclimation, the heterotrophic microalgae complete photoautotrophic conversion and salinity acclimation.
Example 2
The microalgae subjected to photoautotrophic conversion and salinity acclimation in example 1 were subjected to algal cell density of 1X 105Measuring cell/mL, adding microalgae with a certain volume into an outdoor culture tail water treatment tank, wherein the volume of the culture tail water treatment tank is 200m3Aerating and mixing with prawn intensive culture effluent, culturing, adding BG11 nutritive salt except nitrogen and phosphorus nutritive salt, i.e. MgSO4·7H20-375mg/L、CaCl2·2H2O-180mg/L、Na2CO3180mg/L, 30mg/L of citric acid, 30mg/L of ferric citrate and 5-1mL/L of trace element solution A.
Initial values of nitric acid nitrogen, nitrous acid nitrogen, ammonia nitrogen and total inorganic phosphorus in the discharged water measured according to a method of ocean monitoring Specification (GB17378.4-2007) are 12.77, 6.89, 7.02 and 4.27mg/L respectively.
Through the biological purification effect of the chlorella pyrenoidosa, the result shows that the removal rates of ammonia nitrogen, total inorganic phosphorus, nitric acid nitrogen and nitrous acid nitrogen are 92.18%, 24.73%, 3.68% and 1.42% respectively on day 9; on day 18, total inorganic phosphorus, nitric acid nitrogen and nitrous acid nitrogen removal rates were 57.56%, 69.63% and 68.78%, and chlorella pyrenoidosa cell biomass increased 39.25 times.
In summary, heterotrophic microalgae were photoautotrophic converted and salinity acclimated at 1 × 105The algae cell density inoculation treatment of the prawn culture discharge water can effectively remove nitrogen and phosphorus, realize the rapid amplification of algae biomass, and achieve the purification effect of the discharge wastewater and the supply of primary productivity to a certain extent.
Example 3
The chlorella pyrenoidosa that had been photoautotrophic converted and salinity acclimated in example 1 was cultured at a cell density of 5X 105Measuring cell/mL, measuring microalgae with a certain volume, and addingIn the outdoor culture tail water treatment tank, the volume of the culture tail water treatment tank is 200m3Aerating and mixing with prawn intensive culture effluent, culturing, adding BG11 nutritive salt except nitrogen and phosphorus nutritive salt, i.e. MgSO4·7H20-375mg/L、CaCl2·2H2O-180mg/L、Na2CO3180mg/L, 30mg/L of citric acid, 30mg/L of ferric citrate and 5-1mL/L of trace element solution A.
Initial values of nitric acid nitrogen, nitrous acid nitrogen, ammonia nitrogen and total inorganic phosphorus in the discharged water measured according to a method of ocean monitoring Specification (GB17378.4-2007) are 12.77, 6.89, 7.02 and 4.27mg/L respectively.
Through the biological purification effect of algae cells, the result shows that the removal rates of ammonia nitrogen, total inorganic phosphorus, nitric acid nitrogen and nitrous acid nitrogen are 95.57%, 33.89%, 2.79% and 0.35% respectively on day 9; on day 18, total inorganic phosphorus, nitric acid nitrogen and nitrous acid nitrogen removal rates were 67.99%, 80.62% and 51.18%, and chlorella pyrenoidosa cell biomass increased 7.98 times.
In summary, heterotrophic microalgae were photoautotrophic converted and salinity acclimated at 5 × 105The algae cell density inoculation treatment of the prawn culture discharge water can effectively remove nitrogen and phosphorus, realize the rapid amplification of algae biomass, and achieve the purification effect of the discharge wastewater and the supply of primary productivity to a certain extent.
Example 4
The chlorella pyrenoidosa that had been photoautotrophic-converted and salinity-acclimated in example 1 was cultured at a cell density of 10X 105Measuring cell/mL, adding microalgae with a certain volume into an outdoor culture tail water treatment tank, wherein the volume of the culture tail water treatment tank is 200m3Aerating and mixing with prawn intensive culture effluent, culturing, adding BG11 nutritive salt except nitrogen and phosphorus nutritive salt, i.e. MgSO4·7H20-375mg/L、CaCl2·2H2O-180mg/L、Na2CO3180mg/L, 30mg/L of citric acid, 30mg/L of ferric citrate and 5-1mL/L of trace element solution A.
Initial values of nitric acid nitrogen, nitrous acid nitrogen, ammonia nitrogen and total inorganic phosphorus in the discharged water measured according to a method of ocean monitoring Specification (GB17378.4-2007) are 12.77, 6.89, 7.02 and 4.27mg/L respectively.
Through the biological purification effect of algae cells, the result shows that the removal rates of ammonia nitrogen, total inorganic phosphorus, nitric acid nitrogen and nitrous acid nitrogen are 97.97%, 36.23%, 24.40% and 0.91% respectively on day 9; on day 18, total inorganic phosphorus, nitric acid nitrogen and nitrous acid nitrogen removal rates were 81.37%, 74.88% and 50.85%, with a 4.06-fold increase in algal cell biomass.
In summary, heterotrophic microalgae were photoautotrophic converted and salinity acclimated at 10 × 105The algae cell density inoculation treatment of the prawn culture discharge water can effectively remove nitrogen and phosphorus, realize the rapid amplification of algae biomass, and achieve the purification effect of the discharge wastewater and the supply of primary productivity to a certain extent.
Claims (5)
1. A method for purifying tail water of marine culture by heterotrophic microalgae is characterized in that the microalgae is Chlorella pyrenoidosa, the Chlorella pyrenoidosa cultured by heterotrophic fermentation is centrifuged and washed for 2 times, photoautotrophic conversion and salinity domestication are carried out, the microalgae after photoautotrophic conversion and salinity domestication are inoculated into the tail water of the marine culture for culture, and the inoculation density is 1 multiplied by 105-106cell/mL, which enables microalgae cells to cooperatively purify culture discharge wastewater in the culture process;
the photoautotrophic conversion method comprises the following steps: diluting the washed microalgae by 1000 times according to the volume ratio, adding a complete BG11 culture medium, wherein the culture conditions are that the temperature is 25-28 ℃, the illumination intensity is 5000-;
the salinity domestication method comprises the following steps: the salinity of the heterotrophic fermentation medium is 10 per mill, and NaCl with corresponding weight is added to the required final concentration according to the salinity increased by 5 per mill every day.
2. A method according to claim 1, characterised in that the final concentration of the salinity condition corresponds to the concentration of the wastewater to be treated.
3. The method of claim 1, wherein the photoautotrophic conversion simultaneously performs salinity acclimation of the heterotrophic microalgae.
4. The method according to claim 1, characterized in that the method for heterotrophic fermentative culture of chlorella pyrenoidosa comprises: inoculating chlorella pyrenoidosa algae liquid which is subjected to aseptic culture in logarithmic growth phase into a bioreactor, wherein a fermentation culture medium comprises 30g/L of glucose, 25g/L of yeast extract, 0.5g/L of potassium dihydrogen phosphate, 0.5g/L of magnesium sulfate, 0.5g/L of sodium citrate, 130 mg/L of vitamin B, 630 mg/L of vitamin B, 125 mg/L of vitamin B and 2mg/L of biotin, the pH value is 6.5, the temperature is 30 ℃, and the continuous fermentation culture is carried out for 120 hours.
5. The method as claimed in claim 1, wherein the final concentration of each nutrient salt added in the microalgae cultivation cooperative purification cultivation wastewater discharge process is MgSO4·7H20-375mg/L、CaCl2·2H2O-180mg/L、Na2CO3180mg/L, 30mg/L of citric acid, 30mg/L of ferric citrate and 1mL/L of trace element A5 solution.
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