CN109897785B - Stable culture method for indoor simulation of water bloom - Google Patents
Stable culture method for indoor simulation of water bloom Download PDFInfo
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Abstract
The invention discloses a stable culture method for indoor simulated water bloom, which takes wild natural water as inflow raw water and wild unseparated water bloom microalgae as an algae source, has low cost of nutritive salt and short culture period, and the cell concentration of the obtained microalgae is 6.68 multiplied by 104~512.17×104The cells/mL range, the Chl a reaches 11.24-197.39 mu g/L, the Chl a reaches the water bloom definition standard, the obtained microalgae is mixed microalgae, the photosynthetic activity is high, the field water bloom is simulated to a certain extent, a novel method for stably culturing the mixed microalgae indoors is realized, and a stable algae source is provided for the subsequent water bloom formation mechanism and control measure research. Furthermore, the continuous and stable culture of the indoor microalgae is realized by combining the chemostat culture device. The method can be used for stable culture of the mixed microalgae, can also be used for research on the competitive relationship among different microalgae, and has good application prospect.
Description
Technical Field
The invention relates to the technical field of algae culture methods, in particular to a stable culture method for simulating water bloom indoors.
Background
Algal Blooms (Algal Blooms) refers to a natural ecological phenomenon of mass propagation of algae in fresh water bodies, and isRichness of water body Nutrition foodIs characterized in that after the wastewater containing a large amount of nitrogen and phosphorus in the domestic and industrial and agricultural production enters the water body,blue (B) Algae (Saccharum sinensis Roxb.)(also called asCyanobacteriaComprises thatMicrocystis、Anabaena、Oscillatoria algae、Nostoc algaeBlue ball algae,Hair weedsEtc.),Green algae、Diatom algaeAnd the water body is blue or green after mass propagation. Water bloom is an environmental problem faced by many countries around the world at present, and is rich in many nutrientsIn the raised lake or reservoir, algae are abnormally propagated, accumulated in a local lake area or reservoir area and decomposed at high temperature to form malodor; particularly, when algae are accumulated in a large amount near the intake of the water source, the water quality of the water source may be deteriorated, which endangers the safety of water supply. Therefore, when the water bloom treatment method is researched, the technology of simulating the water bloom, predicting and forecasting the water bloom is developed, the decision-making capability of the environmental management department can be improved, and the relevant departments can be facilitated to take emergency measures to deal with the water bloom pollution in time.
At present, the group microalgae isolated from the field are cultured indoors and often exist in the form of single cells. However, under the natural conditions in the field, most microalgae usually exist in the form of groups, so that the physiological performance of the microalgae cultivated indoors and the microalgae cultivated in the field has a certain difference, and great errors often exist when the microalgae is used for researching the water bloom formation mechanism and treatment measures. On one hand, the change of field environmental conditions is large, and the culture environment similar to that of the field is difficult to simulate in a laboratory; on the other hand, the field microalgae are mixed algae, zooplankton and bacteria exist in an environment medium, and the indoor research adopts sterile pure culture and lacks interaction such as reciprocity, symbiosis, competition, cooperation and the like among species or in-species. In addition, microalgae are greatly influenced by environmental conditions, and the physiological state of algae cells is difficult to capture during tests, so that the traditional culture system has limitations in developing research on microalgae physiological characteristics, metabolite transfer and nutrient salt utilization. However, the traditional microalgae culture adopts intermittent culture, the environmental conditions in the system change along with the change of the culture time after the microalgae usually passes through a lag phase, a log phase, a stationary phase and a decline phase, so that the physiological characteristics of the microalgae also change along with the change of the culture time, and the microalgae culture is mainly used for pure algae culture. The field bloom is composed of a plurality of microalgae, bacteria and zooplankton exist around the field bloom, if the field bloom is cultured by adopting a traditional microalgae culture method, the physiological characteristics, the types and the quantity of the microalgae in the system are constantly changed, the obtained microalgae have randomness, and the field bloom can not provide an algae source with consistent physiological state, type and quantity for the subsequent experiment. Therefore, there is a need in the art for a hybrid microalgae cultivation method, which realizes stable cultivation similar to field algal bloom by simulating cultivation of algal bloom, and has an important role in providing stable algae sources for exploring formation mechanism or treatment measures of algal bloom.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a stable culture method for simulating water bloom indoors, and solves the problems that the obtained microalgae obtained by the conventional indoor culture method is single in type, and the physiological performance of the microalgae is different from that of natural water microalgae, so that the research result error of the microalgae is large and the physiological state of algae cells is difficult to capture.
In order to solve the technical problems, the invention adopts the following technical scheme: a stable culture method for simulating water bloom indoors comprises the following steps:
1) collecting microalgae from water body of wild water bloom outbreak, removing impurities visible to naked eyes, and sealing for later use;
2) collecting a field natural (water bloom outbreak) water body, filtering the water body by a 0.45-micron membrane, and adding nitrate and/or phosphate to adjust the TN of the natural water body to be 0.5-2.5 mg/L and the TP to be 0.05-0.25 mg/L so as to obtain a microalgae culture solution; inoculating the microalgae obtained in the step 1) into the microalgae culture solution in a sterile environment, placing the microalgae culture solution in an illumination incubator for expanding culture for 3-5 days to adapt to an indoor culture environment, increasing biomass, centrifuging the expanded microalgae, discarding supernatant, rinsing with the microalgae culture solution for 2-5 times, and removing impurities and background nutritive salts;
3) inoculating the microalgae obtained in the step 2) into a chemostat to be cultured for 10-15 days, and obtaining the stable mixed microalgae.
Further, the chemostat adopted by the invention comprises a reaction kettle which is arranged in a closed manner, a bloom culture chamber which is arranged at an interval with the kettle wall is arranged in the reaction kettle, a closed water bath cavity is formed between the bloom culture chamber and the kettle wall, the bottom and the top of the kettle wall are respectively provided with a water bath port which is communicated with the water bath cavity, and the two water bath ports are connected with a constant-temperature water cooler. Adopt above-mentioned structure, the circulating water that the constant temperature cold water machine will return from the water bath chamber heats or cools off to the constant temperature after, carries to the water bath chamber again, and the circulating water that carries to the water bath chamber returns to the constant temperature cold water machine again after carrying out heat exchange with the interior little algae culture solution of wawter bloom culture room and heats or cools off, and reciprocating circulation plays the constant temperature effect to the interior little algae culture solution of wawter bloom culture room.
A stirring mechanism for stirring culture solution is also arranged in the bloom culture chamber; the bottom of the reaction kettle is provided with an air vent communicated with the bloom culture chamber, and the air vent is connected with an air pumping mechanism through a pipeline; the culture device also comprises a sample introduction unit for conveying nutrients, a light source unit and an algae liquid backflow unit.
The sample introduction unit comprises a sample introduction bottle which is connected with a sample introduction pump through a pipeline and extends to the inside of the reaction kettle and is positioned above the liquid level of the microalgae culture solution of the bloom culture chamber. Thus, when the phosphorus and nitrogen nutrition of the microalgae culture solution in the water bloom culture chamber is not suitable, the biomass of the microalgae is adjusted or the dominant species of the microalgae is changed by adjusting the nutrient substances in the sample feeding bottle.
The algae liquid reflux unit comprises a reflux bottle and a filter bottle which are arranged in a closed manner, and the reflux bottle and the filter bottle are communicated through a water system filter membrane; an active backflow pipeline and a passive backflow pipeline are connected to the backflow bottle, one end of the active backflow pipeline extends into the position below the liquid level of the backflow bottle, the other end of the active backflow pipeline is connected with a backflow peristaltic pump, the backflow peristaltic pump is connected into the reaction kettle through the active backflow pipeline and located on the liquid level of the microalgae culture solution in the bloom culture chamber, one end of the passive backflow pipeline extends into the position below the liquid level of the microalgae culture solution in the bloom culture chamber, and the other end of the passive backflow pipeline is connected to the position above the liquid level of the backflow bottle; the filter flask is connected with a water outlet pump through a pipeline, and the pipeline extends into the position below the liquid level of the filter flask.
Adopt above-mentioned structure, when little algae culture solution volume increase sinks passive return line, too much little algae culture solution can follow passive return line and discharge from the water bloom culture room and directly get into the backward flow bottle, communicate with water system filtration membrane between backward flow bottle and the filter flask, water system filtration membrane only allows the water in the backward flow bottle to pass through, get into the filter flask, the drainage gets into out the water-jug through the delivery pump in the filter flask, make to have the liquid level difference between filter flask and the backward flow bottle, promote the water in the backward flow bottle to get into the filter flask, and concentrated algae liquid remains in the backward flow bottle, concentrated algae liquid in the backward flow bottle can flow back to the water bloom culture room through the reflux pump.
Preferably, the stirring mechanism comprises a stirring shaft which is vertically arranged in the bloom culture chamber in a rotatable manner and a stirring piece which is radially arranged along the stirring shaft, wherein the upper end of the stirring shaft extends out of the reaction kettle and is connected with an output shaft of a motor, so that the stirring shaft rotates under the control of the motor. Furthermore, three groups of double-blade blades are uniformly distributed on the stirring piece along the circumferential direction of the stirring shaft; still further, the blades are 45-degree inclined blades, and each group of blades mutually form a 60-degree angle.
By adopting the structure, the blade surface and the moving direction form a certain inclination angle, so when the blade moves, the blade also has axial shunting besides horizontal circulation, the stirring area and the stirring efficiency are increased, and the uniform mixing of gas, liquid and algae liquid is ensured.
Preferably, the pore diameter of the water-based filtration membrane is 0.45 to 2 μm.
Preferably, the bloom culture chamber of the reaction kettle also comprises plastic beads for mixing the bloom, the density of the plastic beads is close to that of water, and the plastic beads can be suspended in the culture solution. Therefore, the plastic beads rotate uniformly in a suspension state along with the rotation of the stirring blades, the stirring area and the stirring efficiency are increased, and the uniform mixing of gas, liquid and algae liquid is ensured. The rotating speed of the stirring piece, the diameter of the plastic balls and the number of the plastic balls are determined according to the size of the chemostat, for example, when the chemostat with the effective volume of 1L is adopted, the stirring speed is set to be 150-250 r/min, the number of the plastic balls is 100-200, and the particle size of the plastic balls is 10-50 mm.
Preferably, the pipelines in the culture device are all silica gel tubes, and the silica gel tubes are connected with the bottles in a rubber sleeve mode. Therefore, the airtightness of the reaction kettle is ensured, the overlarge pressure in the reaction kettle is avoided, and the safety of the culture device is ensured.
Preferably, one end of the passive return pipe extends into the algae liquid level in the bloom culture chamber, and the liquid level does not exceed the opening of the passive return pipe. Like this, when the indoor algae liquid level of water bloom cultivates risees and does not cross the opening of passive back flow, too much algae liquid passes through pressure action and gets into the backward flow bottle along passive back flow, and the algae liquid after the concentration can follow the initiative back flow and flow back to the water bloom cultivates the room in the backward flow bottle, avoids making little algae biomass low excessively because of algae liquid runs off at the excessive speed, also avoids algae liquid concentration to change too greatly simultaneously, produces great influence to little algae growth and reproduction.
Preferably, the air pumping mechanism comprises an air pump, the air pump is connected with the air vent through a pipeline, the air pump is further provided with a gas flowmeter, and the gas conveying pipe is provided with an air filtering head. Thus, sterile air is introduced into the bloom culture chamber from the air vent through the air filter head, and the ventilation flow is controlled through the gas flowmeter.
Preferably, the light source unit comprises led lamps uniformly distributed along the top and the periphery of the reaction kettle. Like this, control illumination, the dark of cultivateing the in-process through time switch to the day night environment of simulation natural water.
Preferably, the nitrate is one or more of sodium nitrate, potassium nitrate, ammonium nitrate and calcium nitrate; the phosphate is one or more of potassium dihydrogen phosphate, sodium phosphate, potassium hydrogen phosphate.
Preferably, the culture conditions in the chemostat are that the temperature is 20-30 ℃, the illumination intensity is 2000-2500 lux, the light-dark ratio is 12h:12h, and the ventilation volume is 100 ml/min.
Preferably, the dilution ratio of the chemostat is 0.1-0.6, the reflux ratio is 0.5-2.0, the stirring speed of the stirring mechanism is 150-250 r/min, and the dilution ratio is the water inlet speed (ml · d · min)-1) The ratio of the volume (ml) of the culture system algae liquid to the reflux rate (ml. d)-1) With the rate of water entry (ml. d)-1) The ratio of. Thus, the reflux ratio and the dilution ratio can be regulated and controlled automatically according to the concentration of the cultured microalgae and the specific requirements of nutrient salts in the culture system, the microalgae with different specific growth rates can be obtained, other microalgae can be eliminated according to the specific growth rate range of the target microalgae, and the microalgae can be used as the culture medium for culturing microalgae in the culture systemSo as to obtain the desired water bloom microalgae. The range of the dilution rate and the reflux ratio is suitable for the specific growth rate of the existing algae, and the growth and even death of the algae can be influenced by too large or too small of the dilution rate and the reflux ratio.
Compared with the prior art, the invention has the following beneficial effects:
1. the cell concentration of the microalgae obtained by the method is 6.68 multiplied by 104~512.17×104The cells/mL range, the Chl a reaches 11.24-197.39 μ g/L, all reach the water bloom definition standard. The microalgae is mixed microalgae, has high photosynthetic activity and no obvious difference between the physiological performance of the microalgae and the microalgae in a natural water body, simulates field bloom to a certain extent, realizes a new method for stably culturing the mixed microalgae indoors, also improves the correctness of results, and simultaneously provides a stable algae source for the research of a subsequent bloom formation mechanism and control measures.
2. The invention takes the field natural water as the water inlet raw water, takes the field unseparated water bloom microalgae as the algae source, has low cost of nutritive salt and short culture period, and is combined with the chemostat culture device to realize the continuous and stable culture of the indoor microalgae. The method can be used for stable culture of mixed microalgae, can also be used for research on the competitive relationship among different microalgae, and has good application prospect.
3. According to the invention, the novel chemostat structure is designed, microalgae is cultured, and gas-liquid and algae-liquid are uniformly mixed in a mode of ventilating through the stirring mechanism, the plastic balls and the bottom; meanwhile, the algae liquid is concentrated and refluxed through the algae liquid reflux system, so that the problem of too low microalgae biomass caused by too high loss rate is avoided. In addition, the regulation and control of the flow in the sample inlet system, the aeration system, the algae liquid reflux system and the sample outlet system are realized through the plurality of peristaltic pumps, the peristaltic pumps are not interfered with each other, the peristaltic pumps are regulated according to the growth condition of the microalgae in the reaction kettle, the device is suitable for the culture of different nutrient salt concentrations and different water blooms, and the problems that the variety of the obtained microalgae is single, the physiological performance of the obtained microalgae is different from that of the microalgae in a natural water body, the physiological state of algae cells is difficult to capture and the error of the microalgae research result is large in indoor culture are solved.
Drawings
FIG. 1 is a schematic diagram of the chemostat of the present invention;
FIG. 2 is a schematic view of the structure of the stirring member of FIG. 1;
FIG. 3 is the Fv/Fm curve of the maximum light energy conversion efficiency of microalgae in example 3 under different illumination times during stabilization;
FIG. 4 is a graph showing the density and ratio of microalgae in example 3 when stabilized.
Detailed Description
The present invention will be described in further detail with reference to examples.
Example 1
A stable culture method for simulating water bloom indoors comprises the following steps:
1) collecting 1000 mL of water with depth of 0.5m from the surface layer from the microcystis bloom lake, removing macroscopic impurities, and sealing for later use.
2) Collecting natural water, filtering with 0.45 μm membrane, measuring TN and TP concentration, and adding 0.5mg/mLNaNO3(in terms of N), 0.05mg/ml KH2PO4Regulating TN to 0.5mg/L and TP to 0.05mg/L (calculated by P) and pure water, finally sterilizing at high temperature, and cooling to obtain a microalgae culture solution;
inoculating the microalgae obtained in the step 1) into 2L of the microalgae culture solution in a sterile environment, placing the microalgae culture solution in a light incubator for propagation for 4d, wherein the culture conditions are that the temperature is 25 ℃, the light is 2000-2500 lux, the light-dark ratio is 12h:12h, and the ventilation is 100 ml/min; and then centrifuging the expanded microalgae at the rotating speed of 5000rpm for 5min, discarding the supernatant, and rinsing the microalgae with the microalgae culture solution for 2-3 times.
3) Inoculating the microalgae obtained in the step 2) into a chemostat in a sterile environment, and culturing at the temperature of 25 ℃, the illumination of 2000-2500 lux, the light-dark ratio of 12h:12h and the ventilation of 100 ml/min. Wherein the dilution ratio of the chemostat is 0.2, the stirring speed is 150r/min, the reflux ratio is 1:1, the effluent is 0.7Q (Q is the inflow rate), and the effluent is 0.3Q. After 11d of culture, stabilization was achieved. Thus obtaining the stable mixed microalgae.
The water bloom stabilizing characteristic: the mixed microalgae obtained by the identification of the embodiment mainly comprises 7 genus microalgae, wherein the 3 genus of cyanophyta and the 4 genus of chlorophyta. The cell density of algae is 24.08 × 104cells/mL, wherein the blue algae accounts for more than 92 percent, and the formed water bloom mainly containing the blue algae reaches the water bloom definition standard, namely the method disclosed by the invention is adopted to simulate the field water bloom indoors.
Referring to fig. 1 and 2, the chemostat structure adopted by the invention comprises a reaction kettle 1 which is arranged in a closed manner, wherein a bloom culture chamber 11 which is arranged at an interval with the kettle wall is arranged in the reaction kettle 1, a closed water bath cavity 12 is formed between the bloom culture chamber 11 and the kettle wall, the bottom and the top of the kettle wall are respectively provided with a water bath port which is communicated with the water bath cavity 12, and the two water bath ports are connected with a constant temperature water cooler 7. By adopting the structure, the circulating water returned from the water bath cavity 12 is heated or cooled to a constant temperature by the constant temperature water cooler 7, and then is conveyed to the water bath cavity again, and the circulating water conveyed to the water bath cavity and the algae liquid in the bloom culture chamber are subjected to heat exchange and then are returned to the constant temperature water cooler again for heating or cooling, and are circulated repeatedly, so that the algae liquid in the bloom culture chamber has a constant temperature effect.
A stirring mechanism 4 for stirring culture solution is also arranged in the bloom culture chamber 11; the bottom of the reaction kettle is provided with an air vent 5 communicated with the bloom culture chamber 11, and the air vent 5 is connected with an air pumping mechanism through a pipeline; the culture device also comprises a sample introduction unit for conveying nutrients, a light source unit and an algae liquid backflow unit.
The sample introduction unit comprises a sample introduction bottle 21, wherein the sample introduction bottle 21 is connected with a sample introduction pump 22 through a pipeline and extends to the inside of the reaction kettle 1 and is positioned above the liquid level of the algae liquid in the bloom culture chamber 11. Thus, when the phosphorus and nitrogen nutrition of the algae liquid in the water bloom culture chamber is not suitable, the biomass of the microalgae is adjusted or the dominant algae species are changed by adjusting the nutrient substances in the sample feeding bottle.
The algae liquid reflux unit comprises a reflux bottle 62 and a filter bottle 64 which are arranged in a sealing way, and the reflux bottle 62 and the filter bottle 64 are communicated through a water system filter membrane 63; an active backflow pipeline and a passive backflow pipeline are connected to the backflow bottle 62, one end of the active backflow pipeline extends into the position below the liquid level of the backflow bottle 62, the other end of the active backflow pipeline is connected with a backflow peristaltic pump 61, the backflow peristaltic pump 61 is connected into the reaction kettle through the active backflow pipeline, one end of the passive backflow pipeline extends into the position below the liquid level of the bloom culture chamber 11, and the other end of the passive backflow pipeline is connected to the position above the liquid level of the backflow bottle 62; the filter bottle 64 is connected with a water outlet pump 65 through a pipeline, and the water outlet pipe extends into the position below the liquid level of the filter bottle 64.
By adopting the structure, when the volume of the algae liquid is increased, the excessive algae liquid is discharged from the bloom culture chamber along the passive return pipe and directly enters the return bottle, the return bottle is communicated with the filter bottle through the water system filter membrane, the water system filter membrane only allows water in the return bottle to pass through and enter the filter bottle, filtered water in the filter bottle enters the water outlet bottle through the water outlet pump, so that a liquid level difference exists between the filter bottle and the return bottle, the water in the return bottle is promoted to enter the filter bottle, the concentrated algae liquid is remained in the return bottle, and the concentrated algae liquid in the return bottle can flow back to the bloom culture chamber through the return pump.
When the device is used, the upper end of the reflux bottle is provided with a water outlet which is connected with the stock discharge bottle 31 through a pipeline and used for collecting excessive algae liquid in the reflux bottle so as to avoid excessive algae liquid in the reflux bottle and excessive pressure; the drain pump 65 is connected to the drain bottle 32 through a pipe for collecting the filtered water discharged through the drain pump 65. Wherein, the stock flask 31 and the drain flask 32 are not hermetically arranged with the pipeline.
During implementation, the stirring mechanism comprises a stirring shaft 41 which is vertically arranged in the bloom culture chamber in a rotatable manner and a stirring piece 42 which is arranged along the radial direction of the stirring shaft, wherein the upper end of the stirring shaft extends out of the reaction kettle and is connected with an output shaft of a motor, so that the stirring shaft rotates under the control of the motor. Further, three groups of double-blade blades are uniformly distributed on the stirring piece 42 along the circumferential direction of the stirring shaft 41; still further, the blades are 45-degree inclined blades, and each group of blades mutually form a 60-degree angle.
By adopting the structure, the blade surface and the moving direction form a certain inclination angle, so when the blade moves, the blade also has axial shunting besides horizontal circulation, the stirring area and the stirring efficiency are increased, and the uniform mixing of gas, liquid and algae liquid is ensured.
In practice, the pore diameter of the aqueous filtration membrane 63 is 0.45 to 2 μm.
In practice, the bloom culture chamber of the reaction kettle also comprises plastic beads 8 for mixing the bloom, and the plastic beads can be suspended in the culture solution. Therefore, the plastic beads rotate uniformly in a suspension state along with the rotation of the stirring blades, the stirring area and the stirring efficiency are increased, and the uniform mixing of gas, liquid and algae liquid is ensured.
During implementation, the pipelines in the culture device are all silica gel tubes, and the silica gel tubes are connected with the bottles in a rubber sleeve mode. Therefore, the airtightness of the reaction kettle is ensured, the overlarge pressure in the reaction kettle is avoided, and the safety of the culture device is ensured.
When in implementation, one end of the active return pipeline connected with the reaction kettle is positioned above the liquid level of the bloom culture chamber 11. Like this, when the interior algae liquid level of water bloom culture room rose, too much algae liquid got into the backward flow bottle through passive back flow, then the algae liquid after the concentration in the backward flow bottle can follow the initiative back flow and flow back to the water bloom culture room, avoided making little algae biomass low because of algae liquid runs off at the excessive speed, also avoided simultaneously that algae liquid concentration changes too greatly, produced great influence to the growth and the reproduction of little algae.
When the air pumping mechanism is implemented, the air pumping mechanism comprises an air pump 53, the air pump is connected with the air vent 5 through a pipeline, the air pump is further provided with a gas flowmeter 52, and the gas conveying pipe is provided with an air filter head 51. Thus, sterile air is introduced into the bloom culture chamber from the air vent through the air filter head, and the ventilation flow is controlled through the gas flowmeter.
When the device is implemented, the light source unit comprises led lamps uniformly distributed along the top and the periphery of the reaction kettle. Like this, control illumination, the dark of cultivateing the in-process through time switch, the day night environment of simulation natural water.
Example 2
A stable culture method for simulating water bloom indoors comprises the following steps:
1) collecting 1000 mL of water with depth of 0.5m from the surface layer from the microcystis bloom lake, removing macroscopic impurities, and sealing for later use.
2) Collecting natural water body and passing through 0After 45 μm membrane filtration, the TN and TP concentrations were measured, and 0.5mg/mLNaNO was added thereto3(in terms of N), 0.05mg/ml KH2PO4Adjusting TN to 0.5mg/L and TP to 0.05mg/L (measured by P) and pure water, finally sterilizing at high temperature, cooling to obtain a microalgae culture solution, inoculating the microalgae obtained in the step 1) into 2L of the microalgae culture solution in a sterile environment, placing the microalgae culture solution in an illumination incubator for expansion culture for 4 days, wherein the culture conditions are that the temperature is 25 ℃, the illumination is 2000-2500 lux, the light-dark ratio is 12h:12h, and the air is introduced for 100 ml/min. And then centrifuging the expanded microalgae at the rotating speed of 5000rpm for 5min, discarding the supernatant, and rinsing the microalgae with the microalgae culture solution for 2-3 times.
3) Inoculating the microalgae obtained in the step 2) into a chemostat with the structure as described in the example 1 in a sterile environment, and culturing at the temperature of 25 ℃, the illumination of 2000-2500 lux, the light-dark ratio of 12h:12h and the ventilation of 100 ml/min. Wherein the dilution ratio of the chemostat is 0.4, the stirring speed is 150r/min, the reflux ratio is 1:1, the effluent is 0.7Q (Q is the inflow), the effluent is 0.3Q, and the mixture is stabilized after being cultured for 14 days, so that the stable mixed microalgae is obtained.
The water bloom stabilizing characteristic: the mixed microalgae obtained by the embodiment is identified to mainly comprise 9 genus microalgae, wherein the cyanophyta 2 belongs, the chlorophyta 5 belongs and the diatom 2 belongs. The cell density of algae is 6.68 × 104 cells/mL, wherein the blue algae accounts for 44.67%, the green algae accounts for 54.48%, and the diatom accounts for 0.85%, forms blue-green algae bloom which reaches the bloom definition standard, namely the invention simulates field bloom indoors.
Example 3
A stable culture method for simulating water bloom indoors comprises the following steps:
1) collecting 1000 mL of water with depth of 0.5m from the surface layer from the microcystis bloom lake, removing macroscopic impurities, and sealing for later use.
2) Collecting natural water, filtering with 0.45 μm membrane, measuring TN and TP concentration, and adding 0.5mg/mLNaNO3(in terms of N), 0.05mg/ml KH2PO4Regulating TN to 0.5mg/L and TP to 0.05mg/L (calculated as P) and pure water, sterilizing at high temperature, and cooling to obtain microalgae culture solutionInoculating the microalgae obtained in the step 1) into 2L of the microalgae culture solution in a sterile environment, placing the microalgae culture solution in a light incubator for propagation for 4d, wherein the culture conditions are that the temperature is 25 ℃, the light illumination is 2000-2500 lux, the light-dark ratio is 12h:12h, and the ventilation is 100 ml/min. And then centrifuging the expanded microalgae at the rotating speed of 5000rpm for 5min, discarding the supernatant, and rinsing the microalgae with the microalgae culture solution for 2-3 times.
3) Inoculating the microalgae obtained in the step 2) to a chemostat with the structure as described in the example 1 in a sterile environment, and culturing at the temperature of 25 ℃, the illumination of 2000-2500 lux, the light-dark ratio of 12h:12h and the ventilation of 100 ml/min. Wherein the dilution ratio of the chemostat is 0.2, the stirring speed is 150r/min, the reflux ratio is 1:1, the effluent is 0.7Q (Q is the inflow rate), and the effluent is 0.3Q. After 11d of culture, stabilization was achieved.
4) After the microalgae in the step 3) are stabilized, regulating the TN of the inlet water to 2.5mg/L and the TP to 0.25mg/L, and after the microalgae are cultured for 3 days, finding that the biomass of the microalgae is obviously increased, in order to simplify the reactor device, continuously culturing after withdrawing the reflux part device, and culturing for 10 days to be stabilized.
Water bloom characteristics at steady state: the mixed microalgae obtained in the embodiment is identified to mainly comprise genus 5 microalgae, genus Oscillatoria, genus Chlorella, genus Chlamydomonas, genus Navicula and genus Arthrospira, wherein genus Cyanophyta 1, genus Chlorophyta 2 and genus Diatoma 2. The cell density of the algae is 512.17 × 104 cells/mL, wherein the blue algae accounts for 98.44% (figure 3), forms blue algae water bloom taking Oscillatoria as dominant algae, and reaches the water bloom definition standard, namely the invention simulates field water bloom indoors. And the obtained microalgae still has the characteristic of stable growth after the cultured microalgae is subjected to secondary culture.
The photosynthetic activity was measured by forming bloom in this example, and the results are shown in FIG. 4.
As can be seen from FIG. 4, the potential maximum light energy conversion efficiency Fv/Fm of the blue algae during the illumination period of the water bloom formed by the method is within the range of 0.58-0.62, and the potential maximum light energy conversion efficiency Fv/Fm of the blue algae is within the range of 0.63-0.67, which are all higher than those of wild blue algae and green algae. The photosynthetic activity of the formed water bloom is higher, and the culture method does not form light stress on the microalgae and can better simulate the field water bloom phenomenon.
The above description is only exemplary of the present invention and should not be taken as limiting, and any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A stable culture method for simulating water bloom indoors is characterized by comprising the following steps:
1) collecting microalgae from water body with wild water bloom outbreak, removing macroscopic impurities, and sealing for later use;
2) collecting wild natural water, filtering by a 0.45-micron membrane, adding nitrate and/or phosphate, adjusting the total nitrogen and the total phosphorus of the water to be 0.5-2.5 mg/L and 0.05-0.25 mg/L, and sterilizing at high temperature to obtain a microalgae culture solution; inoculating the microalgae obtained in the step 1) into the microalgae culture solution in a sterile environment, placing the microalgae culture solution in an illumination incubator for 3-5 days for propagation, centrifuging the propagated microalgae, removing supernatant, and rinsing with the microalgae culture solution for 2-5 times;
3) inoculating the microalgae obtained in the step 2) into a chemostat to be cultured for 10-15 days to obtain stable mixed microalgae; the cell concentration of the obtained mixed microalgae is 6.68 × 104~512.17×104The cell/mL range is that the chlorophyll A reaches 11.24-197.39 mu g/L;
culturing the microalgae in the step 2) and the step 3) under the conditions that the temperature is 20-30 ℃, the illumination intensity is 2000-2500 lux, the light-dark ratio is 12h:12h, and the ventilation volume is 100 ml/min;
the chemostat comprises a reaction kettle (1) which is arranged in a closed manner, a bloom culture chamber (11) which is arranged at an interval with the kettle wall is arranged in the reaction kettle (1), a closed water bath cavity (12) is formed between the bloom culture chamber (11) and the kettle wall, the bottom and the top of the kettle wall are respectively provided with a water bath port which is communicated with the water bath cavity (12), and the two water bath ports are connected with a constant-temperature water cooler (7); a stirring mechanism (4) for stirring the culture solution is also arranged in the bloom culture chamber (11); the bottom of the reaction kettle is provided with an air vent (5) communicated with the bloom culture chamber (11), and the air vent (5) is connected with an air pumping mechanism through a pipeline; the culture device also comprises a sample introduction unit for conveying nutrients, a light source unit and an algae liquid backflow unit, wherein the sample introduction unit comprises a sample introduction bottle (21), the sample introduction bottle (21) is connected with a sample introduction pump (22) through a pipeline, extends into the reaction kettle and is positioned above the liquid level of the microalgae culture solution in the bloom culture chamber (11);
the algae liquid reflux unit comprises a reflux bottle (62) and a filter bottle (64) which are hermetically arranged, and the reflux bottle (62) and the filter bottle (64) are communicated through a water system filter membrane (63); an active backflow pipeline and a passive backflow pipeline are connected to the backflow bottle (62), one end of the active backflow pipeline extends into the position below the liquid level of the backflow bottle (62), the other end of the active backflow pipeline is connected with a backflow peristaltic pump (61), the backflow peristaltic pump (61) is connected into the reaction kettle through the active backflow pipeline and is located above the liquid level of the microalgae culture solution in the bloom culture chamber (11), one end of the passive backflow pipeline extends into the position of the liquid level of the microalgae culture solution in the bloom culture chamber (11), and the other end of the passive backflow pipeline is connected to the position above the liquid level of the backflow bottle (62); the filter bottle (64) is connected with a water outlet pump (33) through a water outlet pipeline, and the water outlet pipeline extends into the position below the liquid level of the filter bottle (64);
the dilution ratio of the chemostat is 0.1-0.6, the reflux ratio is 1: 0.5-1: 2.0, and the stirring speed of the stirring mechanism is 150-250 r/min.
2. The indoor steady cultivation method for simulating the water bloom according to claim 1, wherein the stirring mechanism comprises a stirring shaft (41) which is rotatably and vertically arranged in the water bloom cultivation chamber and a stirring piece (42) which is radially arranged along the stirring shaft, the upper end of the stirring shaft extends out of the reaction kettle and is connected with an output shaft of a motor, so that the stirring shaft rotates under the control of the motor; three groups of double-blade blades are uniformly distributed on the stirring piece (42) along the circumferential direction of the stirring shaft (41).
3. The method for stably culturing the indoor simulated algal bloom according to claim 2, wherein the leaves are 45 ° inclined leaves, and each set of the leaves are 60 ° each other.
4. The method for stably culturing an indoor simulated algal bloom according to claim 1, wherein the pore diameter of the water system filtration membrane (63) is 0.45 to 2 μm.
5. The method for stably culturing indoor simulated water bloom as claimed in claim 1, wherein the water bloom culture chamber (11) of the reaction kettle further comprises plastic beads (8) for mixing the water bloom, and the density of the plastic beads is close to that of water.
6. The stable cultivation method of indoor simulated water bloom according to claim 1, wherein the air pumping mechanism comprises an air pump (53) connected with a vent (5) through a pipeline, the air pump is further provided with a gas flow meter (52), and the gas delivery pipe is provided with an air filter head (51); the light source unit comprises led lamps uniformly distributed along the top and the periphery of the reaction kettle.
7. The indoor stable cultivation method for simulating water bloom according to claim 1, wherein the nitrate is one or more of sodium nitrate, potassium nitrate, ammonium nitrate and calcium nitrate; the phosphate is one or more of potassium dihydrogen phosphate, sodium phosphate and potassium hydrogen phosphate.
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