CN106635815B - Culture medium and culture method of protodinoflagellate - Google Patents
Culture medium and culture method of protodinoflagellate Download PDFInfo
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Abstract
The invention discloses a proto-polymethacrylic algae culture medium and a culture method, wherein the culture medium comprises the following components in parts by weight: NH (NH)4NO3，20‑100mg；CO(NH2)2(urea), 20-60 mg; KH (Perkin Elmer)2PO4，1‑10mg；NaHCO3，100‑800mg；MnCl2·4H2O 15‑50g；FeC6H5O7·5H2O (ferric citrate), 10-30 mg; VB12, 1X 10‑5‑1×10‑3mg；VB1，50‑200mg；KCl，0.5‑5mg；Na210-15g of EDTA; 0.2-5g (molecular weight 1500-. The culture method of the proto-polymetaphyllum culture medium comprises the following steps: the first stage is as follows: culturing a triangular flask; and a second stage: mineral water bucket cultureAnd (5) nourishing. The invention provides the most suitable nutrient salt formula for culturing the protodinoflagellate, which can improve the yield of protodinoflagellate toxin and reduce the culture cost. And compared with the traditional open cement pool type enlarged culture, the mineral water bucket culture of the protozoite has the advantages of low cost, difficult pollution, easy operation, greatly reduced workload, greatly improved growth speed of the protozoite and shortened culture period.
Description
Technical Field
The invention relates to a formula of an original polymethacrylic algae culture medium and a culture method using a mineral water barrel as a container. Belongs to the field of marine microalgae cultivation.
Background
Due to the characteristics of strong toxicity, unpredictability and the like, the shellfish toxin has serious threats to the health safety of consumers and the offshore ecological safety, and is an ecological disaster and a major marine environment problem which are concerned by the international society in recent years. Azaspiroaric acid toxins (AZA) are novel fat-soluble shellfish toxins which are found in the eight existing shellfish toxins at the latest and have the strongest toxicity, and are important concerns of countries such as European Union, America and Canada due to strong toxicity, high residue and slow metabolism, the limit standard is set to be 160 mu g AZA-1 eq/kg (AZA 1-3), the numerical value is generally lower than that of other shellfish toxins, and the important monitoring is performed in international trade.
Therefore, the detection work of the original domoic acid shellfish toxin becomes necessary guarantee for export trade and consumer safety guarantee. At present, most international standard substance preparation methods are prepared by extracting and purifying positive shellfish samples, and have the main problems that the sample sources are unstable, and the batches have large difference; and the amount of the required positive shellfish sample is large, and the transportation and the extraction and processing pretreatment are complicated. The preparation of the toxin standard substance with toxigenic algae as the source can not only ensure the stable source of the toxin, but also is the best standard substance preparation raw material at present due to less matrix interference. The applicant applies for and deposits toxigenic polymethacryla of azaspiro acid toxin at 2016, 4, 8, and deposits in China center for type microorganism collection, which is classified and named as: azadinium poporum AZDY06 with the biological preservation number of CCTCC NO: m2016181. Has excellent toxin producing character, and the extraction and purification process of toxin is developed primarily. However, continuous expansion culture, nutrient solution circulation and supplement and high-concentration toxigenic algae collection of toxigenic algae become the main bottleneck of the technology.
At present, the original dinoflagellate is cultured by adopting the traditional f/2 culture medium, the growth of algae cells is slow, the cell density is low, the single cell toxin producing capability is weak (40 fg/cell AZA 2), the pollution is easy to occur during the expansion culture, and the centrifugal collection consumes time. Therefore, there is an urgent need to develop a method for the expanded culture of toxigenic polymethacrylalgae and the efficient collection of toxin preparation raw materials. The traditional method for the expanded culture of the algae cells is open cement pool type culture, and the algae cells are very easy to be polluted. The invention provides an optimal nutrient salt formula for original polymethacrylic algae culture, and mineral water bucket is adopted to introduce CO2The method can culture protodinoflagellate with maximum cell density of 6.5 × 106cells/ml, the highest toxin production (320 fg/cell AZA 2), the whole toxin yield is improved by 6 times, and the work load is greatly reduced by the toxin production algae collected by a sedimentation flocculation way.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a culture medium and a culture method of protodinoflagellate.
In order to solve the technical problems, the invention adopts the technical scheme that:
the proto-microalgae culture medium provided by the invention comprises the following components in parts by weight:
NH4NO3，20-100mg；CO(NH2)2，20-60mg；KH2PO41-10 mg; bicarbonate, 100-800 mg; MnCl2·4H2O，15-50g； FeC6H5O7·5H2O，10-30mg；VB12，1×10-5-1×10-3mg；VB1，50-200mg；KCl，0.5-5mg；Na210-15g of EDTA; 0.2-5g of chitosan oligosaccharide with the molecular weight of 1500-2000Da seawater is 1000 mL.
Preferably, the proto-microalgae culture medium provided by the invention comprises the following components in parts by weight:
NH4NO3，30mg；CO(NH2)2，40mg；KH2PO45 mg; bicarbonate, 500 mg; MnCl2·4H2O 26g、FeC6H5O7·5H2O 20mg；VB12，1×10-4mg；VB1，100mg；KCl，3mg；Na212.4g of EDTA, 1g of chitosan oligosaccharide with the molecular weight of 1500-2000Da and 1000mL of seawater.
Further, the bicarbonate of the present invention is selected from one or more of sodium bicarbonate, potassium bicarbonate, calcium bicarbonate and ammonium bicarbonate. Preferably, the bicarbonate is selected from sodium bicarbonate.
The invention also provides an improved formula of the culture medium, which is characterized by further comprising 1-3mg of amino acid on the basis of the culture medium. The amino acids are selected from a combination of polar amino acids (hydrophilic amino acids).
The invention also provides a preparation method of the proto-polymethacrylic culture medium, which comprises the following steps:
1) sterilizing seawater for later use;
2) the culture medium of claim 1, wherein the culture medium is prepared by sequentially adding the culture medium to sterilized seawater in the order and dosage, and stirring for 5 min.
The invention provides an enlarged culture method of protodinoflagellate by utilizing the culture medium provided by the invention, which comprises the following steps:
the first stage is as follows: culturing a triangular flask; the seawater is filtered by 0.45 μm fiber membrane and sterilized at high temperature, and has salinity of 30-35; after adding the culture medium components, adjusting the pH to 7.9 +/-0.3; indoor culture, controlling the temperature by an air conditioner, and controlling the temperature to be 20 +/-3 ℃; lighting of the fluorescent tube, light intensity of 5000-: 12 h;
culturing with 3000 plus 5000mL Erlenmeyer flask with working volume of 3/5, inoculating algae in logarithmic phase; inoculation density 2X 104cell/mL-5×104cell/mL; shaking the bottle 6 times a day; the cell density of algae reaches 2 × 105cell/mL-3×105Transferring the cell/mL into the next stage for culture;
and a second stage: mineral water barrel culture; culturing in 18L mineral water bucket, and sterilizing the mineral water bucket by sodium hypochlorite to sterilize seawater; adding sodium hypochlorite containing 20 mg/kg of available chlorine into per cubic meter of seawater, adding sterilized seawater containing sodium hypochlorite into mineral water bucket, aerating for 30 min, stopping aeration, sterilizing for 3-5 hr, rinsing with sterilized seawater for 3 times, and determining that no residual chlorine is available;
the seawater is sterilized seawater filtered by 0.45 μm fiber membrane, has salinity of 30-35, and is added with 8L culture medium components, and the bottle mouth is sealed with sterilizing sealing membrane; introducing compressed air containing 5% carbon dioxide into the air stone, maintaining pH at 7.9 + -0.3, culturing indoors, controlling temperature at 20 + -3 deg.C, continuously irradiating with 6000 + -1000 LUX on both sides for 15 hr to 9 hr; inoculation density 2X 103cell/mL-3×103cell/mL; adding 5L culture solution every three days, culturing for 7-9 days until the density of algae solution reaches 1.0 × 106-6.5×106cell/ml, the algae liquid is brown yellow, and about 5L of culture solution is collected; and continuously adding 5L of culture solution every three days, keeping sealing, and performing circulating culture and continuous collection.
The invention has the beneficial effects that:
at present, the main problems of the primary large-scale culture of the protodinoflagellate are slow growth of algae cells, long multiplication time and easy pollution during the enlarged culture. The proper culture medium and culture expanding container are one of the important factors for realizing the high-density culture of the protozobium.
The previously used medium was not supplemented with sodium bicarbonate, but instead the traditional f/2 medium was used to culture prototheca. The technical scheme of the invention shows that the microalgae can utilize free CO in the air2And also efficiently utilize HCO in the culture solution3 -As a carbon source. Addition of NaHCO3Not only shortens the doubling time of the original dinoflagellate, but also prolongs the growth time of the algae. 0.5g/L NaHCO was added3The culture medium has more comprehensive and balanced nutrition, can greatly improve the growth speed of the original dinoflagellate, and can improve the yield by 150 percent to the maximum extent. And culturing Prorocentrum donghaiense with mineral water bucket method, and adding 0.5g/L NaHCO3The maximum cell density of proto-polymethacrylic algae can reach 6.5 × 10 under the culture condition5compared with the traditional glass conical flask container culture and traditional cement pond culture methods, the cell/ml culture method is limited by volume and materials, can culture 25L of algae liquid at one time, has low requirements on the environment by the materials, and can ensure that algae seeds are not polluted.
The invention provides the most suitable nutrient salt formula for culturing the original dinoflagellate, which can improve the yield of the original dinoflagellate and reduce the culture cost; by increasing Na2The content of EDTA, and chitosan oligosaccharide with proper proportion is added to promote the division and growth of the protodinoflagellate; mineral water barrel is adopted to lead CO to2The mode of compressing air is combined with KH with proper proportion2PO4The buffer solution has the effects of neutralizing the damage of secondary metabolite generated in the growth process of proto-polymethacrylic algae to the culture solution, maintaining the optimum pH, and regulating CO2Can promote photosynthesis of algae cells. The maximum cell density of the algae cultured by the method can reach 6.5 multiplied by 106cells/ml, highest toxin production (320 fg/cell AZA 2), and 6-fold improvement in overall toxin production.
Compared with the traditional open cement pool type enlarged culture, the mineral water bucket culture of the protozoite has the advantages of low cost, difficult pollution, easy operation, greatly reduced workload, greatly improved growth speed of the protozoite and shortened culture period.
In a word, the prorocentrum donghaiense cultured by using the optimized culture medium and adopting the mineral water bucket method has the advantages of high growth speed, short production period, high yield, low cost, easy operation, difficult pollution, easy control, flexibility, diversity, safety and reliability.
Detailed Description
Example 1
This implementationThe proto-polydatin culture medium is prepared by the following formula: NH (NH)4NO3，30mg；CO(NH2)2，40mg；KH2PO4，5mg；NaHCO3，500mg； MnCl2·4H2O 26g、FeC6H5O7·5H2O 20mg；VB12，1×10-4mg；VB1，100mg；KCl，3mg；Na212.4g of EDTA, 1g of chitosan oligosaccharide with the molecular weight of 1500-2000Da and 1000mL of seawater.
The preparation method of the proto-microalgae culture medium comprises the following steps:
1) sterilizing seawater for later use;
2) and sequentially adding the culture medium into sterilized seawater according to the sequence and the dosage in the formula of the culture medium, and then fully stirring for 5 minutes to inoculate for use.
Example 2
The embodiment provides a method for culturing protodinoflagellate, which comprises the following steps:
the first stage is as follows: culturing a triangular flask; the seawater is filtered by 0.45 μm fiber membrane and sterilized at high temperature, has salinity of 30-35, and is added with culture medium components to adjust pH to 7.9 + -0.3; indoor culture, controlling the temperature by an air conditioner, and controlling the temperature to be 20 +/-3 ℃; lighting of the fluorescent tube, light intensity of 5000-: 12 h;
culturing with 3000 plus 5000mL Erlenmeyer flask with working volume of 3/5, inoculating algae in logarithmic phase; inoculation density 2X 104cell/mL-5×104cell/mL; shaking the bottle 6 times a day; the cell density of algae reaches 2 × 105cell/mL-3×105Transferring the cell/mL into the next stage for culture;
and a second stage: mineral water barrel culture; culturing in 18L mineral water bucket, and sterilizing with sodium hypochlorite to obtain sterilized mineral water bucket. Adding sodium hypochlorite containing 20 mg/kg of available chlorine into per cubic meter of seawater, adding sterilized seawater containing sodium hypochlorite into mineral water bucket, aerating for 30 min, stopping aeration, sterilizing for 3-5 hr, rinsing with sterilized seawater for 3 times, and determining that no residual chlorine is available;
the seawater is sterilized seawater filtered by 0.45 μm fiber membrane, has salinity of 30-35, and is added with 8L culture medium components, and the bottle mouth is sealed with sterilizing sealing membrane. Introducing compressed air containing 5% carbon dioxide into the air stone, maintaining pH at 7.9 + -0.3, culturing indoors, controlling temperature at 20 + -3 deg.C, continuously irradiating with 6000 + -1000 LUX on both sides for 15 hr: 9 hr. Inoculation density 2X 103cell/mL-3×103cell/mL; adding 5L culture solution every three days, culturing for 7-9 days, and allowing the density of algae solution to reach 1.0 × 106-6.5×106cell/ml, the algae liquid is brown yellow, and about 5L of culture liquid is collected. And continuously adding 5L of culture solution every three days, keeping sealing, and performing circulating culture and continuous collection.
The toxigenic algae density reaches 1.6 × 10 on day 76cell/ml, continuously culturing until day 9, the density reaches 6.5 × 106cell/ml, and no pollution phenomenon in microscope observation. Detecting the pH value of the culture solution to be 8.1, ensuring the culture condition to be in a good state, and continuously adding the sterilized culture solution for continuous culture. 50mL of the culture medium was collected and the algal toxin content was determined as (320 fg/cell AZA 2). Collecting 5L culture solution, settling for 6h, centrifuging, collecting algae cells, placing in a sample bag, and preparing AZA toxin standard.
On the next day, culture medium was taken to obtain algal cells, and the density of algal cells was found to be 6.2X 106cell/ml, and no pollution phenomenon in microscope observation. Detecting the pH value of the culture solution to be 8.0, ensuring the culture condition to be in a good state, and continuously adding the sterilized culture solution for continuous culture. 50mL of the culture medium was collected and the algal toxin content was determined as (316 fg/cell AZA 2). Collecting 5L culture solution, settling for 6h, centrifuging, collecting algae cells, placing in a sample bag, and preparing AZA toxin standard.
Until 30 days after inoculation, the culture solution is taken to obtain the algae cells, and the density of the algae cells is found to be 5.8 multiplied by 106cell/ml, and no pollution phenomenon in microscope observation. Detecting the pH value of the culture solution to be 7.8, ensuring the culture condition to be in a good state, and continuously adding the sterilized culture solution for continuous culture. 50mL of the culture was collected and the algal toxin content was determined (307 fg/cell AZA 2). Collecting 5L culture solution, settling for 6 hr, centrifuging, collecting algae cells, placing in sample bag, and preparingPreparing AZA toxin standard product.
Example 3 comparative experiment
The culture method described in example 2 and the culture method of the prior art were applied using the medium described in example 1 and using a conventional f/2 medium, comparing the culture effect of the different media and culture methods:
test 1: the culture effect of the culture medium and the culture method of the invention is as follows:
as shown in example 2, the highest amount of collected toxigenic algae was 6.5X 10 using the culture medium and the culture method of the present invention6cells/ml, single cell with high toxicity yield, the highest toxic yield (320 fg/cell AZA 2).
Test 2: culture Medium of the prior art and culture method of the present invention
The culture method of the present invention is applied using a conventional f/2 medium, and mainly comprises:
the first stage is as follows: culturing a triangular flask; the seawater is filtered by 0.45 μm fiber membrane and sterilized at high temperature, has salinity of 30-35, and is added with culture medium components to adjust pH to 7.9 + -0.3; indoor culture, controlling the temperature by an air conditioner, and controlling the temperature to be 20 +/-3 ℃; lighting of the fluorescent tube, light intensity of 5000-: 12 h;
culturing in 5L Erlenmeyer flask with working volume of 3/5, inoculating algae strain in logarithmic phase; inoculation density 2X 104cell/mL-5×104cell/mL; shaking the bottle 6 times a day; the cell density of algae reaches 2 × 105cell/mL-3×105Transferring the cell/mL into the next stage for culture;
and a second stage: mineral water barrel culture; culturing in 18L mineral water bucket, and sterilizing with sodium hypochlorite to obtain sterilized mineral water bucket. Adding sodium hypochlorite containing 20 mg/kg of available chlorine into per cubic meter of seawater, adding sterilized seawater containing sodium hypochlorite into mineral water bucket, aerating for 30 min, stopping aeration, sterilizing for 3-5 hr, rinsing with sterilized seawater for 3 times, and determining that no residual chlorine is available;
the seawater is sterilized by filtering with 0.45 μm fiber membrane, has salinity of 30-35, and has bottleneck after adding 8L culture medium componentSealing with a sterilizing sealing film. Introducing compressed air containing 5% carbon dioxide into the air stone, maintaining pH at 7.9 + -0.3, culturing indoors, controlling temperature at 20 + -3 deg.C, continuously irradiating with 6000 + -1000 LUX on both sides for 15 hr: 9 hr. Inoculation density 2X 103cell/mL-3×103cell/mL；
The result shows that the highest density of toxigenic algae reaches 0.8 multiplied by 106cells/ml, highest toxicity yield was (92 fg/cell AZA 2). The results of comparative experiment 1 show that the amount of toxigenic algae and the highest toxigenic yield are both reduced. It can be seen that the medium is the main factor affecting the amount of virus produced.
Test 3: culture medium of the invention and prior art culture method
The culture method in the prior art comprises the following steps: placing sterilized seawater into a 5L triangular flask, placing the flask into an incubator, wherein the temperature is 20 ℃, the illumination intensity is 60%, and the light-dark ratio is 12 h: and (4) 12 h. Continuously culturing until the toxigenic algae cell is larger than 1.0 × 106cells/ml, collecting algae liquid.
The result shows that the highest density of toxigenic algae reaches 2.0 multiplied by 106cells/ml, highest toxicity yield (245 fg/cell AZA 2). The result of comparative experiment 1 shows that the amount of produced toxigenic algae is reduced by 3 times, and the maximum yield of toxigenic algae is also reduced by 1.3 times. Because the existing culture method is limited by culture conditions, the maximum culture time is 40d, and then the pH value of the culture solution is too low, the toxigenic algae die and subsides, and the requirement of continuous culture cannot be met.
Test 4: prior art culture medium and prior art culture method
The result of using the conventional f/2 culture medium and the culture method of the prior art shows that the highest density of toxigenic algae reaches 5.5 multiplied by 105cells/ml, highest toxin yield was (47 fg/cell AZA 2). The results of comparative experiment 1 show that the amount and the toxic yield of the toxigenic algae are low and the continuous culture cannot be performed. After a maximum growth period of 30d, subculture is required. Therefore, the growth period is longer, and the yield of the AZA2 toxin is low. Can not meet the aim of preparing large-batch standard products.
Example 4
This example provides a proto-microalgae culture medium, which has the following formula: NH (NH)4NO3，30mg；CO(NH2)2，40mg；KH2PO4，5mg；NaHCO3，500mg； MnCl2·4H2O 26g、FeC6H5O7·5H2O 20mg；VB12，1×10-4mg；VB1，100mg；KCl，3mg；Na212.4g of EDTA, 1g of chitosan oligosaccharide with the molecular weight of 1500-2000Da, 1000mL of seawater,
on the basis of the above-mentioned culture medium, 3mg of amino acid was also included. The amino acid is selected from the combination of glycine, serine and threonine in polar amino acid, and the proportion of the three is 3:2: 1.
The preparation method of the proto-microalgae culture medium comprises the following steps:
1) sterilizing seawater for later use.
2) And sequentially adding the culture medium into sterilized seawater according to the sequence and the dosage in the formula of the culture medium, and then fully stirring for 5 minutes to inoculate for use.
Using the culture medium modified in this example, and the culture method of example 2, the highest amount of collected toxigenic algae was 9.6X 106cells/ml, single cell with high toxicity yield, the highest toxic yield (365 fg/cell AZA 2).
Claims (5)
1. A proto-microalgae culture medium is characterized by comprising the following components in parts by weight:
NH4NO3，20-100mg；CO(NH2)2，20-60mg；KH2PO41-10 mg; bicarbonate, 100-800 mg; MnCl2·4H2O，15-50g； FeC6H5O7·5H2O，10-30mg；VB12，1×10-5-1×10-3mg；VB1，50-200mg；KCl，0.5-5mg；Na210-15g of EDTA; 0.2-5g of chitosan oligosaccharide with the molecular weight of 1500-2000Da and 1000mL of seawater.
2. The proto-microalgal culture medium of claim 1, comprising the following components in weight:
NH4NO3，30mg；CO(NH2)2，40mg；KH2PO45 mg; bicarbonate, 500 mg; MnCl2·4H2O 26g、FeC6H5O7·5H2O 20mg；VB12，1×10-4mg；VB1，100mg；KCl，3mg；Na212.4g of EDTA, 1g of chitosan oligosaccharide with the molecular weight of 1500-2000Da and 1000mL of seawater.
3. The proto-microalgal culture medium according to claim 1 or 2, wherein the bicarbonate is selected from the group consisting of sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, ammonium bicarbonate, or a combination thereof.
4. The method for preparing a prototheca medium according to claim 1, comprising the steps of:
1) sterilizing seawater in the culture medium formula for later use;
2) sequentially adding the rest components in the culture medium formula into the sterilized seawater obtained in the step 1) according to the sequence and the dosage, and then fully stirring for 5 minutes to obtain the culture medium for inoculation and use.
5. The method for culturing toxigenic dinoflagellate is characterized by comprising the following steps:
the first stage is as follows: culturing a triangular flask; the seawater is filtered by 0.45 μm fiber membrane and sterilized at high temperature, and has salinity of 30-35; adjusting the pH to 7.9 ± 0.3 after addition of the media components of claim 1; indoor culture, controlling the temperature by an air conditioner, and controlling the temperature to be 20 +/-3 ℃; lighting of the fluorescent tube, light intensity of 5000-: 12 h;
culturing with 3000 plus 5000mL Erlenmeyer flask with working volume of 3/5, inoculating algae in logarithmic phase; inoculation density 2X 104cell/mL-5×104cell/mL; shaking the bottle 6 times a day; the cell density of algae reaches 2 × 105cell/mL-3×105Transferring the cell/mL into the next stage for culture;
and a second stage: mineral water barrel culture; culturing in 18L mineral water bucket, and sterilizing the mineral water bucket by sodium hypochlorite to sterilize seawater; adding sodium hypochlorite containing 20 mg/kg of available chlorine into per cubic meter of seawater, adding sterilized seawater containing sodium hypochlorite into mineral water bucket, aerating for 30 min, stopping aeration, sterilizing for 3-5 hr, rinsing with sterilized seawater for 3 times, and determining that no residual chlorine is available;
the seawater is sterilized seawater filtered by 0.45 μm fiber membrane, has salinity of 30-35, and is added with 8L culture medium components, and the bottle mouth is sealed with sterilizing sealing membrane; introducing compressed air containing 5% carbon dioxide into the air stone, maintaining pH at 7.9 + -0.3, culturing indoors, controlling temperature at 20 + -3 deg.C, continuously irradiating with 6000 + -1000 LUX on both sides for 15 hr to 9 hr; inoculation density 2X 103cell/mL-3×103cell/mL; adding 5L culture solution every three days, culturing for 7-9 days until the algae solution is brown yellow, and collecting about 5L culture solution; and continuously adding 5L of culture solution every three days, keeping sealing, and performing circulating culture and continuous collection.
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| 蒋保周.氮磷营养盐对腹孔环胺藻（Azadinium poporum）生长及产毒水平的影响.《中国优秀硕士学位论文全文数据库工程科技I辑》.2016,第1-70页，尤其是第22页表2-1，第34页中间段，第43页第18行，第42页5-6行，第20-21页2.2.1 腹孔环胺藻的培养部分. * |
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