CN109566393A - For extracting the cultural method of the seaweed of polysaccharide - Google Patents

For extracting the cultural method of the seaweed of polysaccharide Download PDF

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Publication number
CN109566393A
CN109566393A CN201811532157.3A CN201811532157A CN109566393A CN 109566393 A CN109566393 A CN 109566393A CN 201811532157 A CN201811532157 A CN 201811532157A CN 109566393 A CN109566393 A CN 109566393A
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culture
polysaccharide
seaweed
frustule
pond
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王斌
张建设
夏立萍
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Zhejiang Ocean University ZJOU
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Zhejiang Ocean University ZJOU
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G33/00—Cultivation of seaweed or algae
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05D—INORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C; FERTILISERS PRODUCING CARBON DIOXIDE
    • C05D3/00—Calcareous fertilisers
    • C05D3/02—Calcareous fertilisers from limestone, calcium carbonate, calcium hydrate, slaked lime, calcium oxide, waste calcium products
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F5/00—Fertilisers from distillery wastes, molasses, vinasses, sugar plant or similar wastes or residues, e.g. from waste originating from industrial processing of raw material of agricultural origin or derived products thereof
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management

Abstract

Rich two stages of phosphorus culture and phosphate starvation culture are carried out the present invention is provided to extract the cultural method of the seaweed of polysaccharide, including to frustule；Phosphate starvation cultivation stage first uses shaking flask culture 24-36h, is then transferred to frustule and continues to cultivate in culture pond；Inoculating rotifer culture solution in the culture pond, wheel animalcule culture solution are inoculated with by the 3-5% of Algae culture solution；And during cultivating, intermittent illumination is carried out to culture pond using artificial light source, light intensity uses, and daily illumination 3-5h, each light interval 30min are primary.The present invention cultivates algae using phosphate fertilizer plant's waste water, can low-cost processes phosphate fertilizer plant waste water, turn waste into wealth simultaneously, turn harm into good, comprehensive utilization benefit is high；It is high using alga cells polyoses content obtained by this method culture.

Description

For extracting the cultural method of the seaweed of polysaccharide
Technical field
The invention belongs to algae to cultivate field, and in particular to for extracting the cultural method of the seaweed of polysaccharide.
Background technique
Pollution caused by the rapid development of Phosphate Fertilizer Industry is on the rise in recent years, has caused the extensive concern of people.Money Material display, 1 ton of calcium magnesium phosphate of every production will discharge 40 tons of waste water.The waste water fluoro-containing concentration being discharged in phophatic fertilizer production process is up to 120mg/L.Fluorine is a kind of pair of human body and animal body and the harmful element of environment, and scientific research personnel investigates display, phosphate fertilizer plant worker, family Belong to and 300 meters of surrounding within resident den tal fluorosis recall rate be 8.4%, children, teenager Geng Gao, be 36%, fluoride pollution can also draw The fluorosis of bone that bone density increase and bone trabecula thickening, grid increase, structural fuzzy etc. are symptom is played, to blood of human body and nerve There is adverse effect.Calcium superphosphate is most common phosphate fertilizer, and raw material is phosphorite or apatite and 62-70% sulfuric acid, production reaction Process is as follows: releasing pernicious gas hydrogen fluoride and ocratation during dissolution phosphorite, uses in the condensing unit of sealing Water washing is decomposed into as nontoxic silicic acid and silicofluoric acid.The waste water being discharged from this process is colorless and transparent, weakly acidic.It crosses The production of calcium phosphate is often combined with the production of prodan, therefore is eluted with water and exhaust gas and recycled prodan, just at For the method for wastewater treatment.Prodan can become a kind of valuable byproduct through filtering, washing, desiccation.Waste water if you need into The processing of one step, then can be filtered by lime stone filter bed, or be neutralized with agstone or lime slurry, and the waste water after neutralization passes through Precipitating is discharged, main sodium chloride-containing and calcium in sediment.The method handles waste water investment greatly, such as the sewage treatment of middle-size and small-size phosphate fertilizer plant Equipment investment is up to 100,000 yuan or more, and year running cost also needs 9 Wan Duoyuan, takes up a large area, and troublesome in poeration, treatment effect is by day Gas, temperature and production technology are affected, complex process, and the waste residue stacking problem after lime neutralizes also is difficult to solve, and are easy to make At secondary pollution.What is more, and untreated waste water is directly discharged into river or city by bright ditch or secret tunnel by many phosphate fertilizer plants City's sewer causes to seriously affect to environmental and human health impacts, can also cause the secondary pollutions such as water eutrophication.
In order to solve the deficiency of current technology, the present invention provides a kind of method using phosphate fertilizer plant's waste water cultivation algae, both Energy low-cost processes phosphate fertilizer plant waste water, and the biological product for having important food value can be obtained, turn waste into wealth, turns harm into good, it is comprehensive It is good to close utilization benefit, sewage treatment equipment investment and running expense can be greatly reduced, achieve many things at one stroke, be phosphate fertilizer plant's wastewater treatment Optimal selection.
The Chinese invention patent of existing Publication No. CN 105695349A, discloses a kind of phosphate starvation culture and improvement yeast The cultural method of cell intracellular trehalose, belongs to field of microbial fermentation.This method is trained using molasses as carbon source in initial medium Support yeast cells, increase cellular biomass, after yeast cells reaches certain biomass, be transferred in without phosphorus culture medium or by Add carbon source, nitrogen source according to certain proportion stream, but phosphorus source be not added, carry out phosphate starvation culture, with this increase by 180 grams of cellular biomass/ Liter or more while, greatly improve the accumulation of yeast cells intracellular trehalose, accumulation is up to 13% or more.This method technique letter List, operation is easy, with short production cycle, production cost is low, and Trehalose in Yeast content height can be improved it and prepare the resistance to of active dry yeast Storage characteristic.However, the invention only optimizes nutrient media components, there is no propose specifically to be conducive to improve trehalose The training method or operation of content.
Summary of the invention
The purpose of the present invention is to provide the cultural methods of the seaweed for extracting polysaccharide, cultivate algae using phosphate fertilizer plant's waste water Class, can low-cost processes phosphate fertilizer plant waste water, turn waste into wealth simultaneously, turn harm into good, comprehensive utilization benefit is high；It is trained using this method It is high to support gained alga cells polyoses content.
The technical solution that the present invention is taken to achieve the above object are as follows:
In Initial stage of culture, the culture medium culture algae being made up of suitable carbon source, nitrogen source, phosphorus source and inorganic salts promotes It grows, to promote the synthesis of its cellular biomass；Phosphorus source uses phosphate fertilizer plant's waste water, can low-cost processes phosphate fertilizer plant it is useless Water is turned waste into wealth simultaneously；After algae cell reaches certain biomass, transfers them in without phosphorus culture medium and continues to cultivate, Mainly stablize the accumulation of biomass in frustule at this time, while keeping its normal growth.
For extracting the cultural method of the seaweed of polysaccharide, comprising the following steps:
Rich phosphorus culture: Initial stage of culture, the parts by weight of each component in culture medium are as follows: waste water 100-120 parts of phosphate fertilizer plant, magnesium salts 0.5-0.8 parts, 0.5-1.2 parts of sylvite, 1.5-3.5 parts of nitrate, 1-3 parts of bicarbonate；Wherein, magnesium salts, sylvite, nitrate and Bicarbonate is conventional containing Mg2+、K+、NO3-And HCO3-Inorganic salts；And make Mg2+Concentration is 5-12mg/L, K+Concentration For 520-680mg/L, NO3-Concentration is 450-900mg/L, HCO3-Concentration is 5-13mg/L；These carbon sources, nitrogen source, phosphorus source and nothing Machine salt is all substance necessary to algal grown, wherein Mg2+Polysaccharide synthase can be promoted to activate；K+Maintain and adjust frustule Inside and outside normal osmotic pressure, while promoting the transport of other nutriments；Nitrogen source is the primary raw material of protein synthesis；HCO3-It mentions For growing necessary carbon source, while also providing the life condition of alkalinity；Phosphorus, must as the structural unit for constituting polysaccharide in phosphate It is indispensable；Participate in photosynthesis: photosynthetic phosphorylation must have phosphorus participation, and the transport of photosynthate also be unable to do without phosphorus；Culture Initial stage promotes frustule eubolism and synthesizes the biological substances such as polysaccharide by element necessary to supplement algal grown, is polysaccharide The accumulation of equal biomass provides good basis；
Phosphate starvation culture: late stage of culture carries out phosphate starvation culture to frustule, the parts by weight of each component in culture medium are as follows: 80-100 parts of the beet molasses of sugar content 5-8%, 0.4-0.6 parts of magnesium salts, 0.5-1.0 parts of sylvite, 2-5 parts of nitrate；When algae is thin Born of the same parents' biomass reaches a certain level, and makes algae fast-growth using the culture medium of main carbonaceous sources, and to objects such as the carbohydrates of synthesis Matter carries out stablizing accumulation, it should its eutrophy be avoided to grow；
Polyose extraction: harvest algal cultures are simultaneously dry to generate dried culture；It is ground so that alga cells wall is broken It splits；Dried culture is extracted to generate the extract liquor containing algal polysaccharides, and extract liquor is isolated and purified to obtain algal polysaccharides Extract liquor.
Preferably, doing following pretreatment to phosphate fertilizer plant's waste water of culture algae: it is heavy first to stand phosphate fertilizer plant's waste water 0.5-2h is dropped, floating material, silt, leaf, polybag biggish solid content in equal volume is filtered to remove, sewage is then introduced into cement In pond after exposure 0.1-1 days, quick lime is added in water, alum is added after 5-8h, then stirs sewage, through natural sedimentation, Fermentation, exposure 36-48h, skim the floating materials such as water surface foam, do not stir bottom of pond, take sewage at the middle and upper levels, be packed into lighttight black In color plastic barrel, sealing, 0-5 DEG C of refrigerator or freezer are saved, spare；Wherein, the additional amount of quick lime is every liter of water 0.5-2g, bright The additional amount of alum is every liter of water 1-2.5g.
Preferably, initial-stage culture uses shaking flask culture, condition of culture are as follows: pressing algae/culture medium is 50-80ml/250ml Frustule is collected in inoculation, 25-35 DEG C, 150-170r/min culture 36-48h, centrifugation.
Preferably, phosphate starvation cultivation stage, first uses shaking flask culture culture 24-36h, frustule is then transferred to culture Continue to cultivate in pond；The culture pond is mixed brick pond body, 20 meters long, 15 meters wide, 1.2 meters high, east-west, to receive more sun Illumination；One row's glass is installed above culture pond, culture medium is made to be in a kind of semi-closed state, neither blocking sunlight irradiates algae Liquid, carry out photosynthesis, and be avoided that the dust in air, silt, leaf, paper scrap, protozoan and other microorganisms spore Son etc. is polluted into culture solution；During culture, holding culture solution height is 45cm, and cultivation temperature control is at 25-30 DEG C, light It is 3000-12000Lux according to intensity；Small air pump is installed in culture bottom of pond portion, so that culture solution forms microfluidic, stimulates algae Cell fast-growth；Culture solution was replaced in every seven days.
Preferably, being done the wash completely before culture pond inoculation frustule, and carry out ultraviolet lamp or high pressure nitrogen disinfection Then compound iodic disinfecting liquid re-disinfection 20-30min is added in 5-10min.
Preferably, compound iodic disinfecting liquid is the mixed solution of 1-20g/L containing plant extracts and available iodine 2-8g/L；It is multiple Square iodic disinfecting liquid the preparation method comprises the following steps: by kuh-seng, Cortex Phellodendri, Chinese prickly ash crush after using 35-50% alcohol solution dipping 24-36 it is small When, 250-300 silk cover filtering, filtrate extracts to obtain plant extracts in 105-120 DEG C of distillation；Medicinal iodine is configured to the cream of emulsification Then shape object divides 2-3 times and is added in plant extracts, after the completion of last time is added, add sodium alginate and Sucrose Fatty Acid Ester Fat acid esters stirs 15-20min, until mixed solution uniform surface is homogeneous, obtains compound iodic disinfecting liquid, is sealed in black agents bottle In, it is labelled, it is spare to be placed in 0-3 DEG C of refrigerator；Wherein, the additional amount of sodium alginate and sucrose fatty ester is respectively every liter and disappears Content is 0.05-0.1g and 0.2-0.5g in venom；The special presence of sodium alginate and sucrose fatty ester, facilitates plant The effective component of extract is solubilized into micelle, is separated with iodine formation, the interaction for reducing effective ingredients in plant and iodine (is dropped Influence of the low organic matter to iodine), the stability of thimerosal mixed solution was both kept, but also effective ingredients in plant is mutually assisted with iodine Together；In addition, the emulsification that the two is formed can also enhance plant extracts and iodine to the penetrability of bacteria cell wall and to thin The destructiveness of cell wall enters plant extracts and iodine largely in bacterial cell, and enzyme cofactor and mesostate intracellular is made to escape Out, pathogen metabolic process is caused to be obstructed and bactericidal effect is presented.
Kuh-seng is the root of leguminous plant, and the alkaloid that it contains has significantly gram positive bacteria and gram-negative bacteria Bacteriostasis；Cortex Phellodendri is the skin of rutaceae cork tree or wampee, and antibiotic effective ingredient is berberine, water decoction or alcohol preserved material Gram-positive cocci and gram negative bacilli and Leptospira are all had a strong inhibitory effect in vitro, mechanism is to bacterium The inhibition of breathing and RNA synthesis is related；Chinese prickly ash is the mature pericarp of Rutaceae shrub or dungarunga plant pepper or green pepper；Chinese prickly ash In alkaloid can inhibit DNA isomery enzymatic synthesis, have good selective inhibitory to a variety of moulds, gram positive bacteria； Iodine itself has good bactericidal effect, and the mechanism of action is that free-iodine can directly occur with mycoprotein and bacterium zymoprotein Halogenation, the biological activity for destroying albumen cause microorganism dead, and iodine can effectively kill various microorganisms, while be also good The inactivator of good virus；By compounding plant extracts with iodine, its biocidal efficacies, and safe and non-toxic no dirt can be significantly improved Dye.
Preferably, the inoculating rotifer culture solution in culture pond, wheel animalcule culture solution is inoculated with by the 3-5% of Algae culture solution； And during cultivating, intermittent illumination is carried out to culture pond using artificial light source, light intensity uses 35-60molm-2·s-1, every daylight According to 3-5h, each light interval 30min is primary；It using source stimulating wheel animalcule, is formed and is reunited, and then stimulate alga cells, improved The stress reaction of frustule accelerates growth metabolism and the energy exchange of frustule, may advantageously facilitate the intracellular and extracellular of frustule The synthesis of polysaccharide, while being conducive to improve seaweed growth rate.
The invention has the benefit that
1) present invention using phosphate fertilizer plant's waste water cultivate algae, can low-cost processes phosphate fertilizer plant waste water, turn waste into wealth simultaneously, It turns harm into good, comprehensive utilization benefit is high；It is high using alga cells polyoses content obtained by this method culture；
2) present invention first uses shaking flask culture, then frustule is transferred in culture pond and is cultivated in phosphate starvation cultivation stage； The culture pond is to sterilize by compound iodic disinfecting liquid；The compound iodic disinfecting liquid passes through sodium alginate and sucrose fatty ester for plant Extract is effectively compounded with iodine, and the special presence of sodium alginate and sucrose fatty ester, facilitates having plant extracts Ingredient solubilization is imitated in micelle, is separated with iodine formation, interaction (the i.e. reduction organic matter pair of effective ingredients in plant and iodine is reduced The influence of iodine), the stability of thimerosal mixed solution was both kept, but also effective ingredients in plant is mutually cooperateed with iodine；In addition, two The emulsification that person is formed can also enhance the penetrability of plant extracts and iodine to bacteria cell wall and the destruction to cell wall Property, enter plant extracts and iodine largely in bacterial cell, escape enzyme cofactor and mesostate intracellular, causes cause of disease Body metabolic process is obstructed and bactericidal effect is presented；
3) present invention inoculating rotifer culture solution in culture pond, and intermittent illumination is carried out to culture pond using artificial light source； It using source stimulating wheel animalcule, is formed and is reunited, and then stimulate alga cells, improved the stress reaction of frustule, accelerate frustule Growth metabolism and energy exchange, may advantageously facilitate the synthesis of the intracellular and exocellular polysaccharide of frustule, while be conducive to improve seaweed Growth rate.
Present invention employs above-mentioned technical proposals to provide model essay, compensates for the deficiencies in the prior art, design is reasonable, operation side Just.
Specific embodiment
Present invention is further described in detail with reference to embodiments:
Embodiment 1:
For extracting the cultural method of the seaweed of polysaccharide, comprising the following steps:
(1) rich phosphorus culture: Initial stage of culture, the parts by weight of each component in culture medium are as follows: 100 parts of phosphate fertilizer plant's waste water, magnesium salts 0.5 Part, 0.5 part of sylvite, 1.5 parts of nitrate, 1 part of bicarbonate；Wherein, magnesium salts, sylvite, nitrate and bicarbonate are routine Contain Mg2+、K+、NO3-And HCO3-Inorganic salts；And make Mg2+Concentration is 5mg/L, K+Concentration is 520mg/L, NO3-Concentration is 450mg/L, HCO3-Concentration is 5mg/L；These carbon sources, nitrogen source, phosphorus source and inorganic salts are all substances necessary to algal grown, In, Mg2+Polysaccharide synthase can be promoted to activate；K+It maintains and adjusts normal osmotic pressure inside and outside frustule, while promoting other battalion Support the transport of substance；Nitrogen source is the primary raw material of protein synthesis；HCO3-It provides and grows necessary carbon source, while alkalinity being also provided Life condition；Phosphorus is essential as the structural unit for constituting polysaccharide in phosphate；Participate in photosynthesis: photophosphorylation Effect must have phosphorus participation, and the transport of photosynthate also be unable to do without phosphorus；Initial stage of culture passes through member necessary to supplement algal grown Element promotes frustule eubolism and synthesizes the biological substances such as polysaccharide, provides good basis for the accumulation of the biomass such as polysaccharide；
Wherein, phosphate fertilizer plant's waste water is to pass through following pretreatment: first by phosphate fertilizer plant waste water standing sedimentation 0.5h, being filtered to remove Sewage, is then introduced into cement pit after exposure 0.1 day by floating material, silt, leaf, polybag biggish solid content in equal volume, Quick lime is added in water, alum is added after 5h, then stirs sewage, through natural sedimentation, fermentation, exposure 36h, skims the water surface The floating materials such as foam, do not stir bottom of pond, take sewage at the middle and upper levels, are fitted into lighttight black plastic bucket, sealing, 0 DEG C of refrigerator Or freezer saves, it is spare；Wherein, the additional amount of quick lime is every liter of water 0.5g, and the additional amount of alum is every liter of water 1g；
Culture uses shaking flask culture, condition of culture are as follows: pressing algae/culture medium is 50ml/250ml inoculation, 25 DEG C, 150r/ Min cultivates 36h, and frustule is collected in centrifugation；
(2) phosphate starvation culture: late stage of culture carries out phosphate starvation culture to frustule, the parts by weight of each component in culture medium Are as follows: 80 parts of the beet molasses of sugar content 5%, 0.4 part of magnesium salts, 0.5 part of sylvite, 2 parts of nitrate；When frustule biomass reaches one Determine degree, make algae fast-growth using the culture medium of main carbonaceous sources, and carry out stablizing accumulation to substances such as the carbohydrates of synthesis, Its eutrophy should be avoided to grow；
Culture first uses shaking flask culture culture for 24 hours, is then transferred to frustule and continues to cultivate in culture pond；The culture pond is Mixed brick pond body, it is 20 meters long, 15 meters wide, 1.2 meters high, it is east-west, to receive more solar irradiations；One row is installed above culture pond Glass makes culture medium be in a kind of semi-closed state, and neither blocking sunlight irradiates algae solution, carries out photosynthesis, and be avoided that Spore of dust, silt, leaf, paper scrap, protozoan and other microorganisms in air etc. is polluted into culture solution；Training During supporting, holdings culture solution height is 45cm, and cultivation temperature is controlled at 25 DEG C, intensity of illumination 3000Lux；In culture bottom of pond portion Small air pump is installed, so that culture solution forms microfluidic, stimulates frustule fast-growth；Culture solution was carried out more in every seven days It changes；
Wherein, it before culture pond inoculation frustule, is done the wash completely, and carries out ultraviolet lamp or high pressure nitrogen disinfection Then compound iodic disinfecting liquid re-disinfection 20min is added in 5min；Compound iodic disinfecting liquid is 1g/L containing plant extracts and available iodine The mixed solution of 8g/L；Compound iodic disinfecting liquid the preparation method comprises the following steps: by kuh-seng, Cortex Phellodendri, Chinese prickly ash crush after using 35% ethyl alcohol it is molten Liquid impregnates 24 hours, and 250 silk cover filterings, filtrate extracts to obtain plant extracts in 105 DEG C of distillations；Medicinal iodine is configured to emulsification Then paste divides 2 times and is added in plant extracts, after the completion of last time is added, add sodium alginate and Sucrose Fatty Acid Ester Fat acid esters stirs 15min, until mixed solution uniform surface is homogeneous, obtains compound iodic disinfecting liquid, is sealed in black agents bottle, It is labelled, it is spare to be placed in 0 DEG C of refrigerator；Wherein, the additional amount of sodium alginate and sucrose fatty ester is respectively every liter of thimerosal Middle content is 0.05g and 0.2g；The special presence of sodium alginate and sucrose fatty ester, facilitates the effective of plant extracts Ingredient solubilization separates in micelle with iodine formation, and the interaction for reducing effective ingredients in plant and iodine (reduces organic matter to iodine Influence), the stability of thimerosal mixed solution had both been kept, but also effective ingredients in plant is mutually cooperateed with iodine；In addition, the two The emulsification of formation can also enhance the penetrability of plant extracts and iodine to bacteria cell wall and the destructiveness to cell wall, Enter plant extracts and iodine largely in bacterial cell, escapes enzyme cofactor and mesostate intracellular, cause pathogen Metabolic process is obstructed and bactericidal effect is presented；
Meanwhile the inoculating rotifer culture solution in culture pond, wheel animalcule culture solution are inoculated with by the 3% of Algae culture solution；And it cultivates Period carries out intermittent illumination to culture pond using artificial light source, and light intensity uses 35molm-2·s-1, daily illumination 3h, every time Light interval 30min is primary；It using source stimulating wheel animalcule, is formed and is reunited, and then stimulate alga cells, that improves frustule stress Reaction, accelerates growth metabolism and the energy exchange of frustule, may advantageously facilitate the synthesis of the intracellular and exocellular polysaccharide of frustule, together When be conducive to improve seaweed growth rate.
Embodiment 2:
For extracting the cultural method of the seaweed of polysaccharide, comprising the following steps:
(1) rich phosphorus culture: Initial stage of culture, the parts by weight of each component in culture medium are as follows: 105 parts of phosphate fertilizer plant's waste water, magnesium salts 0.7 Part, 0.8 part of sylvite, 2.3 parts of nitrate, 2 parts of bicarbonate；Wherein, magnesium salts, sylvite, nitrate and bicarbonate are routine Contain Mg2+、K+、NO3-And HCO3-Inorganic salts；And make Mg2+Concentration is 8mg/L, K+Concentration is 625mg/L, NO3-Concentration is 685mg/L, HCO3-Concentration is 9mg/L；
Wherein, phosphate fertilizer plant's waste water is to pass through following pretreatment: first by phosphate fertilizer plant waste water standing sedimentation 1h, being filtered to remove drift Sewage, is then introduced into cement pit after exposure 0.5 day by floating object, silt, leaf, polybag biggish solid content in equal volume, Quick lime is added in water, alum is added after 6h, then stirs sewage, through natural sedimentation, fermentation, exposure 40h, skims water surface bubble The floating materials such as foam, do not stir bottom of pond, take sewage at the middle and upper levels, are fitted into lighttight black plastic bucket, sealing, 2 DEG C of refrigerators or Freezer saves, spare；Wherein, the additional amount of quick lime is every liter of water 1.3g, and the additional amount of alum is every liter of water 1.8g；
Culture uses shaking flask culture, condition of culture are as follows: pressing algae/culture medium is 65ml/250ml inoculation, 32 DEG C, 160r/ Min cultivates 42h, and frustule is collected in centrifugation；
(2) phosphate starvation culture: late stage of culture carries out phosphate starvation culture to frustule, the parts by weight of each component in culture medium Are as follows: 95 parts of the beet molasses of sugar content 6%, 0.5 part of magnesium salts, 0.8 part of sylvite, 3.5 parts of nitrate；When frustule biomass reaches To a certain degree, make algae fast-growth using the culture medium of main carbonaceous sources, and stability product is carried out to substances such as the carbohydrates of synthesis It is tired, it should its eutrophy be avoided to grow；
Culture first uses shaking flask culture culture 28h, is then transferred to frustule and continues to cultivate in culture pond；The culture pond is Mixed brick pond body, it is 20 meters long, 15 meters wide, 1.2 meters high, it is east-west, to receive more solar irradiations；One row is installed above culture pond Glass makes culture medium be in a kind of semi-closed state, and neither blocking sunlight irradiates algae solution, carries out photosynthesis, and be avoided that Spore of dust, silt, leaf, paper scrap, protozoan and other microorganisms in air etc. is polluted into culture solution；Training During supporting, holdings culture solution height is 45cm, and cultivation temperature is controlled at 28 DEG C, intensity of illumination 8000Lux；In culture bottom of pond portion Small air pump is installed, so that culture solution forms microfluidic, stimulates frustule fast-growth；Culture solution was carried out more in every seven days It changes；
Wherein, it before culture pond inoculation frustule, is done the wash completely, and carries out ultraviolet lamp or high pressure nitrogen disinfection Then compound iodic disinfecting liquid re-disinfection 25min is added in 6min；Compound iodic disinfecting liquid is 12g/L containing plant extracts and available iodine The mixed solution of 4.3g/L；Compound iodic disinfecting liquid the preparation method comprises the following steps: by kuh-seng, Cortex Phellodendri, Chinese prickly ash crush after use 40% ethyl alcohol Solution impregnates 30 hours, and 250 silk cover filterings, filtrate extracts to obtain plant extracts in 110 DEG C of distillations；Medicinal iodine is configured to emulsify Paste, be then added three times in plant extracts, last time be added after the completion of, add sodium alginate and sucrose Aliphatic ester stirs 15min, until mixed solution uniform surface is homogeneous, obtains compound iodic disinfecting liquid, is sealed in black agents bottle In, it is labelled, it is spare to be placed in 2 DEG C of refrigerators；Wherein, the additional amount of sodium alginate and sucrose fatty ester is respectively every liter of disinfection Content is 0.08g and 0.35g in liquid；
Meanwhile the inoculating rotifer culture solution in culture pond, wheel animalcule culture solution are inoculated with by the 4% of Algae culture solution；And it cultivates Period carries out intermittent illumination to culture pond using artificial light source, and light intensity uses 45molm-2·s-1, daily illumination 4h, every time Light interval 30min is primary.
Embodiment 3:
For extracting the cultural method of the seaweed of polysaccharide, comprising the following steps:
(1) rich phosphorus culture: Initial stage of culture, the parts by weight of each component in culture medium are as follows: 120 parts of phosphate fertilizer plant's waste water, magnesium salts 0.8 Part, 1.2 parts of sylvite, 3.5 parts of nitrate, 3 parts of bicarbonate；Wherein, magnesium salts, sylvite, nitrate and bicarbonate are routine Contain Mg2+、K+、NO3-And HCO3-Inorganic salts；And make Mg2+Concentration is 12mg/L, K+Concentration is 680mg/L, NO3-Concentration is 900mg/L, HCO3-Concentration is 13mg/L；
Wherein, phosphate fertilizer plant's waste water is to pass through following pretreatment: first by phosphate fertilizer plant waste water standing sedimentation 2h, being filtered to remove drift Sewage, is then introduced into cement pit after exposure 1 day, in water by floating object, silt, leaf, polybag biggish solid content in equal volume Middle addition quick lime adds alum after 8h, then stirs sewage, through natural sedimentation, fermentation, exposure 48h, skims water surface foam Equal floating materials, do not stir bottom of pond, take sewage at the middle and upper levels, be fitted into lighttight black plastic bucket, seal, 5 DEG C of refrigerators or cold Library saves, spare；Wherein, the additional amount of quick lime is every liter of water 2g, and the additional amount of alum is every liter of water 2.5g；
Culture uses shaking flask culture, condition of culture are as follows: pressing algae/culture medium is 80ml/250ml inoculation, 35 DEG C, 170r/ Min cultivates 48h, and frustule is collected in centrifugation；
(2) phosphate starvation culture: late stage of culture carries out phosphate starvation culture to frustule, the parts by weight of each component in culture medium Are as follows: 100 parts of the beet molasses of sugar content 8%, 0.6 part of magnesium salts, 1.0 parts of sylvite, 5 parts of nitrate；When frustule biomass reaches To a certain degree, make algae fast-growth using the culture medium of main carbonaceous sources, and stability product is carried out to substances such as the carbohydrates of synthesis It is tired, it should its eutrophy be avoided to grow；
Culture first uses shaking flask culture culture 36h, is then transferred to frustule and continues to cultivate in culture pond；The culture pond is Mixed brick pond body, it is 20 meters long, 15 meters wide, 1.2 meters high, it is east-west, to receive more solar irradiations；One row is installed above culture pond Glass makes culture medium be in a kind of semi-closed state, and neither blocking sunlight irradiates algae solution, carries out photosynthesis, and be avoided that Spore of dust, silt, leaf, paper scrap, protozoan and other microorganisms in air etc. is polluted into culture solution；Training During supporting, holdings culture solution height is 45cm, and cultivation temperature is controlled at 30 DEG C, intensity of illumination 12000Lux；At culture pond bottom Portion is equipped with small air pump, so that culture solution forms microfluidic, stimulates frustule fast-growth；Culture solution was carried out more in every seven days It changes；
Wherein, it before culture pond inoculation frustule, is done the wash completely, and carries out ultraviolet lamp or high pressure nitrogen disinfection Then compound iodic disinfecting liquid re-disinfection 30min is added in 10min；Compound iodic disinfecting liquid is 20g/L containing plant extracts and available iodine The mixed solution of 2g/L；Compound iodic disinfecting liquid the preparation method comprises the following steps: by kuh-seng, Cortex Phellodendri, Chinese prickly ash crush after using 50% ethyl alcohol it is molten Liquid impregnates 36 hours, and 300 silk cover filterings, filtrate extracts to obtain plant extracts in 120 DEG C of distillations；Medicinal iodine is configured to emulsification Then paste is added three times in plant extracts, after the completion of last time is added, add sodium alginate and Sucrose Fatty Acid Ester Fat acid esters stirs 20min, until mixed solution uniform surface is homogeneous, obtains compound iodic disinfecting liquid, is sealed in black agents bottle, It is labelled, it is spare to be placed in 3 DEG C of refrigerators；Wherein, the additional amount of sodium alginate and sucrose fatty ester is respectively every liter of thimerosal Middle content is 0.1g and 0.5g；
Meanwhile the inoculating rotifer culture solution in culture pond, wheel animalcule culture solution are inoculated with by the 5% of Algae culture solution；And it cultivates Period carries out intermittent illumination to culture pond using artificial light source, and light intensity uses 60molm-2·s-1, daily illumination 5h, every time Light interval 30min is primary.
Comparative example 1:
Ultraviolet lamp or high pressure nitrogen disinfection are only carried out to culture pond, do not use compound iodic disinfecting liquid secondary sterilization；Remaining mistake Journey is consistent with embodiment 2.
Comparative example 2:
Compound iodic disinfecting liquid preparation process does not utilize sodium alginate and sucrose fatty ester, remaining process and embodiment 2 are protected It holds consistent.
Comparative example 3:
Non- inoculating rotifer culture solution, remaining process are consistent with embodiment 2 in culture pond.
Embodiment 4:
Carry out Polyose extraction to the algae strain cultivated in the embodiment of the present invention or comparative example: harvest algal cultures are simultaneously done It is dry to generate dried culture；It is ground so that alga cells wall ruptures；Dried culture is extracted to generate containing algal polysaccharides Extract liquor, and extract liquor is isolated and purified to obtain algal polysaccharides extract liquor；
Wherein, with the following method to algal polysaccharides and the separation of protein: using enzymatic isolation method with Sevag method in conjunction with: tune PH is 6.5, the trypsase of 3% volume of addition, 65 DEG C of heat preservation 6h, boiling water bath 5min enzyme deactivation, after being cooled to room temperature, 4000r/min Centrifugation removal precipitating, is then added chloroform-n-butanol (5:1) of its volume 1/5, acutely shakes 20min, 5000r/min centrifugation 10min divides and removes albuminate, fetches water phase repetitive operation 3 times, can must take off protein liquid, i.e. algal polysaccharides extract liquor；Table 1 is to measure Each polyoses content and recovery rate；Wherein, polyoses content is relative to algae dried culture；It is easy discovery, culture of the present invention obtains The algae arrived is higher containing polyoses content, especially by embodiment 2 and comparative example 3 it is found that inoculating rotifer is trained in Algae culture solution Nutrient solution is conducive to that frustule is stimulated to synthesize polysaccharide.
1 present invention culture algae of table contains polyoses content
Project Polyoses content (mg/g) Polysaccharide extract rate (%)
Embodiment 2 252 98.17
Comparative example 1 208 97.36
Comparative example 2 229 97.83
Comparative example 3 185 96.21
The prior art of routine techniques dawn known to those skilled in the art in above-described embodiment, therefore herein no longer in detail It repeats.
The above embodiments are only used to illustrate the present invention, and not limitation of the present invention, the ordinary skill people of this field Member can also make a variety of changes and modification without departing from the spirit and scope of the present invention.Therefore, all equivalent Technical solution also belong to scope of the invention, scope of patent protection of the invention should be defined by the claims.

Claims (10)

1. the cultural method for the seaweed for extracting polysaccharide, it is characterised in that: carry out rich phosphorus culture to frustule and phosphate starvation is trained It supports；The phosphate starvation cultivation stage, to the wheel animalcule culture solution of frustule culture solution inoculation 3-5%, and using between artificial light source progress Occult light shines, and light intensity uses 35-60molm-2·s-1, daily illumination 3-5h, each light interval 30min are primary.
2. according to claim 1 for extracting the cultural method of the seaweed of polysaccharide, it is characterised in that: the richness phosphorus culture Stage, the parts by weight of each component in culture medium are as follows: waste water 100-120 parts of phosphate fertilizer plant, 0.5-0.8 parts of magnesium salts, sylvite 0.5-1.2 Part, 1.5-3.5 parts of nitrate, 1-3 parts of bicarbonate；The magnesium salts, sylvite, nitrate and bicarbonate are conventional contain Mg2+、K+、NO3-And HCO3-Inorganic salts；And make Mg2+Concentration is 5-12mg/L, K+Concentration is 520-680mg/L, NO3-Concentration For 450-900mg/L, HCO3-Concentration is 5-13mg/L.
3. according to claim 2 for extracting the cultural method of the seaweed of polysaccharide, it is characterised in that: to phosphate fertilizer plant's waste water It does following pretreatment: first by phosphate fertilizer plant waste water standing sedimentation 0.5-2h, being filtered to remove floating material, silt, leaf, polybag etc. Then sewage is introduced into cement pit after exposure 0.1-1 days by the biggish solid content of volume, be added quick lime in water, after 5-8h Alum is added, sewage is then stirred, through natural sedimentation, fermentation, exposure 36-48h, the floating materials such as water surface foam is skimmed, does not stir Dynamic bottom of pond, takes sewage at the middle and upper levels, is fitted into lighttight black plastic bucket, seals, and 0-5 DEG C of refrigerator or freezer save, spare； Wherein, the additional amount of quick lime is every liter of water 0.5-2g, and the additional amount of alum is every liter of water 1-2.5g.
4. according to claim 1 for extracting the cultural method of the seaweed of polysaccharide, it is characterised in that: the richness phosphorus culture Stage, using shaking flask culture, condition of culture are as follows: pressing algae/culture medium is 50-80ml/250ml inoculation, 25-35 DEG C, 150- 170r/min cultivates 36-48h, and frustule is collected in centrifugation.
5. according to claim 1 for extracting the cultural method of the seaweed of polysaccharide, it is characterised in that: the phosphate starvation training The stage of supporting, the parts by weight of each component in culture medium are as follows: 80-100 parts of the beet molasses of sugar content 5-8%, 0.4-0.6 parts of magnesium salts, potassium 0.5-1.0 parts of salt, 2-5 parts of nitrate.
6. according to claim 1 for extracting the cultural method of the seaweed of polysaccharide, it is characterised in that: the phosphate starvation training The stage of supporting first uses shaking flask culture 24-36h, is then transferred to frustule and continues to cultivate in culture pond；The culture pond is mixed brick pond Body, it is 20 meters long, 15 meters wide, 1.2 meters high, it is east-west, to receive more solar irradiations；One row's glass is installed above culture pond, Culture medium is set to be in a kind of semi-closed state, neither blocking sunlight irradiates algae solution, carries out photosynthesis, and be avoided that in air Dust, silt, leaf, paper scrap, protozoan and other microorganisms spore etc. polluted into culture solution；Culture period Between, holding culture solution height is 45cm, and cultivation temperature control is at 25-30 DEG C, intensity of illumination 3000-12000Lux；It is cultivating Bottom of pond portion is equipped with small air pump, so that culture solution forms microfluidic, stimulates frustule fast-growth；Every seven days to culture solution into Row replacement.
7. according to claim 6 for extracting the cultural method of the seaweed of polysaccharide, it is characterised in that: the culture pond connects It before kind frustule, is done the wash completely, and carries out ultraviolet lamp or high pressure nitrogen disinfection 5-10min, compound iodine is then added and disappears Venom re-disinfection 20-30min.
8. according to claim 7 for extracting the cultural method of the seaweed of polysaccharide, it is characterised in that: the compound iodine disappears Venom is the mixed solution of 1-20g/L containing plant extracts and available iodine 2-8g/L；Compound iodic disinfecting liquid the preparation method comprises the following steps: will Kuh-seng, Cortex Phellodendri, Chinese prickly ash crush after use 35-50% alcohol solution dipping 24-36 hours, 250-300 silk cover filtering, filtrate in Plant extracts is extracted to obtain in 105-120 DEG C of distillation；Medicinal iodine is configured to the paste of emulsification, then divides 2-3 times and is added to plant In object extract, after the completion of last time is added, sodium alginate and sucrose fatty ester are added, stirs 15-20min, until Mixed solution uniform surface is homogeneous, obtains compound iodic disinfecting liquid, is sealed in black agents bottle, labelled, is placed in 0-3 DEG C of refrigerator It is spare.
9. according to claim 8 for extracting the cultural method of the seaweed of polysaccharide, it is characterised in that: the sodium alginate Additional amount with sucrose fatty ester be respectively in every liter of thimerosal content be 0.05-0.1g and 0.2-0.5g.
10. the seaweed that the described in any item cultural method cultures for extracting the seaweed of polysaccharide of claim 1-9 obtain, special Sign is: being used for Polyose extraction；Extraction method of polysaccharides are as follows: harvest algal cultures are simultaneously dry to generate dried culture；It is ground It grinds so that alga cells wall ruptures；Dried culture is extracted to generate the extract liquor containing algal polysaccharides, and extract liquor is divided Algal polysaccharides extract liquor is obtained from purifying.
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Application publication date: 20190405