CN113136345B - Method for heterotrophic-autotrophic continuous cultivation of photosynthetic microorganisms, cultivation system and application thereof - Google Patents
Method for heterotrophic-autotrophic continuous cultivation of photosynthetic microorganisms, cultivation system and application thereof Download PDFInfo
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Abstract
The invention relates to the field of microalgae culture, in particular to a method for heterotrophic-autotrophic continuous culture of photosynthetic microorganisms, a culture system and application thereof. The method comprises the following steps: under the first ventilation, photosynthetic microorganisms are sent to a first culture unit for heterotrophic culture, and a first culture solution is obtained; the photosynthetic microorganisms are sent to a second culture unit, and autotrophic culture is carried out under illumination and second ventilation to obtain a second culture solution; in the method, the gas discharged by the first culture unit is used as part or all of the second ventilated gas source, and the gas discharged by the second culture unit is used as part or all of the first ventilated gas source; the photosynthetic microorganisms in the two culture stages are the same or different. The method provided by the invention can strengthen two processes of heterotrophic culture and autotrophic culture simultaneously.
Description
Technical Field
The invention relates to the field of microalgae culture, in particular to a method for heterotrophic-autotrophic continuous culture of photosynthetic microorganisms, a culture system and application thereof.
Background
Microalgae are a low-grade plant which grows in water in a wide variety and has extremely wide distribution, and are a cell factory driven by sunlight, and CO is absorbed by efficient photosynthesis of microalgae cells 2 Converting light energy into chemical energy of carbohydrate such as fat or starch, and releasing O 2 . The microalgae is expected to be used for producing biological energy and chemicals, so that fossil energy can be replaced and CO can be reduced 2 And the purpose of discharging and the like. Microalgae have been widely focused in recent years because of their extremely high productivity. In addition, partial microalgae can grow heterotrophically by utilizing an organic carbon source like bacteria, the growth rate can be increased by tens of times or even tens of times, and the microalgae are mainly cultivated in a fermentation tank. Besides the two nutrition modes, partial microalgae can also grow in a light energy compatible mode, namely, the microalgae can grow by utilizing the light energy and the chemical energy in the organic carbon source at the same time, and the microalgae can grow by utilizing CO 2 And organic carbon sources, the growth rate is higher than autotrophic and heterotrophic. The microalgae light energy mixotrophic culture is also called as mixed nutrient culture, can promote the growth of a plurality of microalgae and the synthesis of protein thereof, and has the advantages of shortening the culture period and realizing the high-density culture of cells, thereby becoming a novel technology for microalgae culture and having important significance in production and economy.
The cost is a core problem of microalgae cultivation, and the microalgae heterotrophic or mixotrophic cultivation process requires a conditional organic carbon source, which is a large part of the cost of the microalgae heterotrophic or mixotrophic cultivation. In order to reduce the growth cost of microalgae, foreign scholars research the influence of glucose, acetic acid, lactic acid, glycerol, glycine and the like on the growth of the micro-confetti, phaeodactylum tricornutum, chlorella pyrenoidosa, spirulina and the like and the accumulation of bioactive substances, and research results show that the growth of the microalgae and the accumulation of the active substances are facilitated by the soluble organic substances with proper concentration. Kirrolia et al (Renewable and Sustainable Energy Reviews 20:642-656) in 2013 compared the cost of microalgae in three different cultivation modes of an open raceway pond, a photobioreactor and a traditional fermenter, and the comparison result shows that the cost of producing grease per unit mass in the three cultivation modes is $ 7.64, $ 24.6 and $ 1.54 respectively, and the cost of producing microalgae biomass per unit mass is $ 1.54, $ 7.32 and $ 1.02 respectively. Although organic carbon sources are required to be added for microalgae cultivation in the fermentation tank, the production cost is not increased, probably because the microalgae grows in the fermentation tank with high efficiency, the microalgae production period is shortened, and other expenses such as labor cost, equipment depreciation cost, occupied land cost and the like for producing microalgae with unit mass are reduced, so that the microalgae production cost is reduced.
The heterotrophic cultivation of microalgae has many advantages, but has a plurality of problems. When algae cells use organic carbon source, a large amount of CO can be generated due to respiration 2 Generally, 36 to 65 percent of organic carbon source can be fixed in the microalgae biomass, and the rest is CO 2 The release of the form of the formula (I) causes the problems of low utilization rate of organic carbon sources, increased carbon emission and the like, and the phenomenon is most prominent in the heterotrophic culture process of microalgae. On the other hand, microalgae consume large amounts of O during heterotrophic growth 2 If the gas exchange cannot be performed in time, the growth of photosynthetic microorganisms can be seriously affected and even the cultivation is failed. Finally, heterotrophically cultivated microalgae have significantly reduced protein and pigment content compared to autotrophically cultivated microalgae, i.e. no light cultivation reduces microalgae biomass quality, and large volume fermenters are very difficult to light.
The information disclosed in the foregoing background section is only for enhancement of understanding of the background of the invention and is for explanation of the shortfall of the prior art that may include information that does not fall within the prior art.
Disclosure of Invention
It is an object of the present invention to perform both heterotrophic and autotrophic processes of photosynthetic microorganisms simultaneously and to enhance both cultivation processes. The second purpose of the invention is to overcome the defects of low quality of microalgae biomass and CO under heterotrophic conditions in the prior art 2 And emission reduction. To this end, a heterotrophic-autotrophic continuous culture is providedA method for culturing photosynthetic microorganism, and its culture system and application are provided.
In order to achieve the above object, the present invention provides in one aspect a method for heterotrophic-autotrophic continuous cultivation of photosynthetic microorganisms, the method comprising: under the first ventilation, photosynthetic microorganisms are sent to a first culture unit for heterotrophic culture, and a first culture solution is obtained; the photosynthetic microorganisms are sent to a second culture unit, and autotrophic culture is carried out under illumination and second ventilation to obtain a second culture solution; in the method, the gas discharged by the first culture unit is used as part or all of the second ventilated gas source, and the gas discharged by the second culture unit is used as part or all of the first ventilated gas source; the photosynthetic microorganisms in the two culture stages are the same or different.
In a second aspect, the present invention provides a system for the continuous cultivation of photosynthetic microorganisms in a heterotrophic-autotrophic serial process, the system comprising: a first cultivation unit for heterotrophic cultivation of photosynthetic microorganisms and a second cultivation unit for autotrophic cultivation of photosynthetic microorganisms, wherein the first cultivation unit comprises a first ventilation device, the second cultivation unit comprises a lighting device and a second ventilation device,
the exhaust port of the first culture unit is communicated with the second air-passing device of the second culture unit, so that the air discharged by the first culture unit is used as an air source of the second air-passing device;
the exhaust port of the second cultivation unit is communicated with the first ventilation device of the first cultivation unit, so that the gas discharged by the second cultivation unit is used as a gas source of the first ventilation device.
In a third aspect the present invention provides a method for producing biomass comprising culturing photosynthetic microorganisms using the method described above and extracting biomass from the resulting photosynthetic microorganisms.
In a fourth aspect, the invention provides a method of producing bioenergy comprising culturing a photosynthetic microorganism using the above method.
The method provided by the invention can effectively strengthen the heterotrophic culture process and the autotrophic culture process simultaneously. In one embodiment of the present invention, the quality of the heterotrophically obtained photosynthetic microorganism can be effectively improved. In particular, the present invention can obtain the following advantages:
(1) The independent culture units are adopted to enable the heterotrophic culture and the autotrophic culture to be carried out independently, so that the heterotrophic culture can be directly converted into the autotrophic culture, the process of converting two culture modes is simplified, the whole process is simpler, the operability is stronger, and the method is suitable for large-scale culture of microalgae.
(2) By collecting and utilizing the exhaust gas of the autotrophic process and providing a gas source for the heterotrophic process, the exhaust gas of the heterotrophic process is collected and utilized and the gas source is provided for the autotrophic process, and meanwhile, the heterotrophic and autotrophic two culture processes are enhanced.
(3) Heterotrophic use may be made of a fermenter of greater volume than the heterotrophic use. The invention is characterized in that CO is rich in the process of heterotrophic culture 2 Is introduced into an autotrophic photobioreactor for the enhanced autotrophic culture of high-concentration microalgae from the heterotrophic process, thereby realizing the CO generated by the breathing action of the autotrophic algae cells on the heterotrophic cultured algae cells 2 In-situ biosolidation of CO in air is overcome 2 The inhibiting effect of the insufficient content on the autotrophic growth of the microalgae cells improves the utilization rate of organic carbon sources of the whole system, reduces the carbon emission, and overcomes the problems of low quality and carbon emission of the microalgae biomass in the large-scale heterotrophic culture process of the microalgae.
Drawings
FIG. 1 is a graph of chlorella growth in a fermenter according to an example of the present invention.
FIG. 2 is a graph of chlorella growth in a tubular reactor in an example of the invention.
FIG. 3 is a graph showing chlorophyll a/dry weight values of Chlorella in a fermenter over time in accordance with an example of the present invention.
FIG. 4 is a graph showing chlorophyll a/dry weight values of Chlorella in a tube reactor over time in an example of the present invention.
FIG. 5 is a graph showing pH change with time of Chlorella in a fermenter according to an example of the present invention.
FIG. 6 is a graph showing pH change with time of chlorella in a tube reactor in the example of the present invention.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
In one aspect, the invention provides a method for heterotrophic-autotrophic continuous cultivation of photosynthetic microorganisms, the method comprising: under the first ventilation, photosynthetic microorganisms are sent to a first culture unit for heterotrophic culture, and a first culture solution is obtained; the photosynthetic microorganisms are sent to a second culture unit, and autotrophic culture is carried out under illumination and second ventilation to obtain a second culture solution; in the method, the gas discharged by the first culture unit is used as part or all of the second ventilated gas source, and the gas discharged by the second culture unit is used as part or all of the first ventilated gas source; the photosynthetic microorganisms in the two culture stages are the same or different.
In a second aspect, the present invention provides a system for the continuous cultivation of photosynthetic microorganisms in a heterotrophic-autotrophic serial process, the system comprising: a first cultivation unit for heterotrophic cultivation of photosynthetic microorganisms and a second cultivation unit for autotrophic cultivation of photosynthetic microorganisms, wherein the first cultivation unit comprises a first ventilation device, the second cultivation unit comprises a lighting device and a second ventilation device,
the exhaust port of the first culture unit is communicated with the second air-passing device of the second culture unit, so that the air discharged by the first culture unit is used as a part or all of air source of the second air-passing device;
the exhaust port of the second cultivation unit is communicated with the first ventilation device of the first cultivation unit, so that the gas discharged by the second cultivation unit is used as a gas source of part or all of the first ventilation device.
The above-described methods and systems of the present invention will be described concurrently, but it should be understood that the methods and systems of the present invention may be used in conjunction with or separately from each other as subject matter of the present invention.
In the present invention, the photosynthetic microorganisms in both heterotrophic and autotrophic processes may be the same or different.
In the present invention, it is understood that heterotrophic-autotrophic continuous cultivation of photosynthetic microorganisms is understood to mean that the heterotrophic cultivation is performed simultaneously with the autotrophic cultivation, such that the gas discharged from the respective cultivation unit can be used in another cultivation unit.
In the present invention, preferably, the first culturing unit and the second culturing unit are connected in series; the photosynthetic microorganisms of the second culture unit are derived from the culture solution after heterotrophic culture of the first culture unit. In this case, after the heterotrophic culture is completed, the culture solution of the heterotrophic culture is partially sent to the second culture unit of the autotrophic culture, and then the rest of the first culture solution can be continuously subjected to the heterotrophic culture in the first culture unit (through additional nutrient solution supplement), so that when the heterotrophic culture is performed in the first culture unit, the second culture unit is also subjected to the autotrophic culture, and then the gas discharged from the first culture unit is used as a gas source for the second ventilation, and the gas discharged from the second culture unit is used as a gas source for the first ventilation, thereby achieving the purposes of improving the quality of photosynthetic microorganisms and reducing the carbon dioxide emission. Wherein preferably the portion of the first culture broth fed to the second culturing unit comprises 70-90% by volume of the total first culture broth. Part of the first culture solution sent to the second culture unit may be diluted or undiluted.
In the system of the present invention, preferably, the culture solution outlet of the first culturing unit is communicated with the algae solution inlet of the second culturing unit, so that the photosynthetic microorganism directly enters the second culturing unit for autotrophic culture after heterotrophic culture in the first culturing unit.
In order to better promote the heterotrophic-autotrophic auxiliary culture of the series, the gas discharged by the second culture unit is preferably collected and compressed as a source of the first aeration. More preferably, the air and gas discharged from the second culturing unit are mixed as the air source of the first ventilation to ensure the ventilation required by the first ventilation.
In this case, in order to be able to use the gas discharged from the second culturing unit, the second culturing unit adopts a closed type photo-bioreactor structure, preferably a tubular, plate and column type photo-bioreactor structure. And the first culture unit is a closed reactor, preferably a fermentation tank structure.
According to the invention, the first culture unit and the second culture unit are communicated through a culture solution conveying pipeline, and a conveying pump can be arranged on the pipeline to facilitate conveying culture solution from the first culture unit to the second culture unit.
For the system of the present invention, in order to enable the gas discharged from the second culturing unit to be more beneficial to the first ventilation, preferably, the gas outlet of the second culturing unit is sequentially connected with the gas collecting device and the pressurizing device, and then is connected with the first ventilation device of the first culturing unit, so that the gas discharged from the second culturing unit is collected and compressed to be used as the gas source of the first ventilation device.
In order to mix the air and the gas discharged by the second cultivation unit as the first ventilation air source, preferably, an air inlet connected with the atmosphere is arranged at the joint of the second cultivation unit and the air collecting device so as to ensure the requirement of the supercharging device on the air quantity. The gas collecting device can be a gas collecting tube, and the pressurizing device can be an air compressor.
According to the invention, the heterotrophic culture stage is not illuminated, but in order to be more beneficial to the autotrophic culture, a two-stage illumination mode is adopted, namely, preferably, the illumination comprises a first-stage illumination and a second-stage illumination, and the light intensity of the first-stage illumination is below 5000lux, preferably 2000-5000lux, more preferably 3000-4000lux;
the light intensity of the second stage illumination is greater than 5000lux, preferably 6000-20000lux, more preferably 10000-15000lux.
According to the invention, the first stage illumination preferably takes more than 10 hours, preferably 10-36 hours, more preferably 12-30 hours, especially 20-24 hours.
According to the present invention, the duration of the second stage illumination is not particularly limited, and the second stage illumination is performed for the remaining cultivation time after the first stage illumination is performed. It is understood that, each time the culture solution from the first culturing unit is sent to the second culturing unit for autotrophic culture, the first-stage illumination is performed first, and then the second-stage illumination is performed.
In the invention, the illumination can be sunlight or artificial light.
According to the present invention, when the diluted first culture solution or microalgae not derived from the first culture solution is subjected to autotrophic culture, the light is preferably sunlight. According to the present invention, in the case of autotrophic culture of the undiluted first culture solution, the illumination is preferably an artificial light source.
According to the invention, when the artificial light source is adopted, the illumination wavelength can be changed in a wider range, and can be partial wavelength light or full wavelength light, and in order to be more beneficial to the growth of photosynthetic microorganisms in the invention, preferably, the first illumination wavelength and the illumination wavelength are 380-780nm, more preferably, the illumination wavelength is 490-460nm and/or 620-760nm, and under the illumination wavelength, photosynthetic microorganism cells can better utilize light energy, and the energy consumption of photosynthetic microorganism cultivation is reduced.
The artificial light source used for the illumination can be an LED light source, in particular a blue light LED light source and a red light LED light source. In order to isolate the water vapor, the artificial light source can be sealed by adopting a transparent material.
According to the present invention, in order to enable microorganisms to better utilize light energy, it is preferable for the artificial light source arrangement of the present invention that the artificial light source has a pitch of 2 to 300mm, preferably 60 to 200mm, in the light direction; or the artificial light source can be directly inserted into the culture solution after being sealed.
According to the invention, the first ventilation is the gas discharged by the second cultivation unit or the mixed gas of the gas discharged by the second cultivation unit and air, and the oxygen content of the gas is higher than that of the air due to the doping of the oxygen generated in the autotrophic cultivation process, so that the heterotrophic cultivation is facilitated. The aeration amount of the first aeration is preferably 0.1-10L/(L.min), preferably 0.2-5L/(L.min), to enable better heterotrophic growth of photosynthetic microorganisms in the first cultivation unit of the invention.
It should be understood that the aeration source used in the first heterotrophic culture may be directly compressed air, and the sterile-processed compressed air may be used.
According to the invention, the gas source adopted by the second ventilation is the gas discharged by the first cultivation unit or the mixture of the gas discharged by the first cultivation unit and the existing gas used for autotrophic cultivation, and the carbon dioxide content of the gas is higher than that of the air due to the doping of the carbon dioxide generated in the heterotrophic cultivation process, so that the autotrophic cultivation is facilitated. Wherein the ventilation amount of the second ventilation is preferably 0.1 to 10L/(L.min), more preferably 0.2 to 5L/(L.min).
According to the present invention, for the system of the present invention, the ventilation means employed for the first ventilation and the second ventilation may be of a ventilation means structure conventionally employed in the art, as long as it can be used for performing the first ventilation and the second ventilation of the present invention.
According to the invention, the heterotrophic culture is carried out under stirring, wherein the stirring speed is preferably 200-500r/min. For this purpose, a stirring structure may be added to the first culturing unit of the system of the invention.
According to the invention, the heterotrophic culture is preferably at 20-35 ℃. The incubation time may vary within a wide range, for example, the time may be 3 to 10 days. The time of heterotrophic culture is herein understood to be the time between each start of culture and the time of sending again to the second culture unit of the autotrophic culture.
According to the invention, the autotrophic culture is preferably carried out at 20-35 ℃. The incubation time can vary within a wide range, for example 3-20 days. The autotrophic cultivation time is herein understood to be both the cultivation time of the photosynthetic microorganisms of each batch and the residence time of the photosynthetic microorganisms in the second cultivation unit of the autotrophic cultivation. That is, the culture solution of the autotrophic culture may be continuously discharged out of the second culturing unit at a certain flow rate.
According to the invention, the first cultivation unit of the invention is provided in a closed arrangement, whereby a sterile heterotrophic cultivation is possible, which requires the supplementation of an organic carbon source. Preferably, the organic carbon source is a saccharide and/or acetate. The saccharide may be, for example, one or more of glucose, fructose, sucrose, maltose, etc. Said may be sodium acetate, for example. More preferably, the organic carbon source is glucose.
Wherein the addition amount of the organic carbon source can be varied within a wide range, and preferably, the addition amount of the organic carbon source is 5-15g/L in the culture system.
Still further, the photosynthetic microorganism is preferably a microalgae, preferably a green algae, more preferably a chlorella.
According to the present invention, in order to achieve a certain sterile environment for heterotrophic culture, the gas used for aeration may be sterile, and in addition, antibiotics may be added to the heterotrophic culture system to prevent bacterial growth. Such antibiotics may be those conventionally employed in the art for controlling the aseptic culture of microorganisms, and may be, for example, one or more of kanamycin, chloramphenicol, streptomycin, gentamicin, vancomycin, azithromycin, and the like. The amount may vary within wide limits, for example from 10 to 65mg/L.
According to the invention, other reagents conventionally used in the art, such as phosphates (e.g. K 2 HPO 4 、Na 2 HPO 4 Etc.). The amounts thereof may be conventional in the art, and the present invention is not particularly limited.
According to the invention, the culture medium used in the culture system of the photosynthetic microorganism preferably has the following composition: k (K) 2 HPO 4 ·3H 2 O：20-50mg/L，NaNO 3 ：1200-2000mg/L，Na 2 CO 3 ：10-30mg/L，MgSO 4 ·7H 2 O：50-90mg/L，CaCl 2 ·2H 2 O:30-50mg/L, citric acid: 1-10mg/L, ferric ammonium citrate: 1-10mg/L, EDTA sodium: 0.5-2mg/L, trace element A5:0.5-2ml/L.
Wherein, the composition of the trace element A5 is preferably as follows: h 3 BO 3 ：2500-3000mg/L，MnCl 2 ·4H 2 O：1500-2000mg/L，ZnSO 4 ·7H 2 O：200-250mg/L，CuSO 4 ·5H 2 O：50-90mg/L，NaMoO 4 ·5H 2 O：350-420mg/L，Co(NO 3 ) 2 ·6H 2 O：20-65mg/L。
In a third aspect the present invention provides a method for producing biomass comprising culturing photosynthetic microorganisms using the method described above and extracting biomass from the resulting photosynthetic microorganisms.
The biomass may be a variety of biomass conventional in the art, and may be, for example, one of oils, proteins, carbohydrates, nucleic acids, pigments, vitamins, growth factors, or any combination thereof.
In a fourth aspect, the invention provides a method of producing bioenergy comprising culturing a photosynthetic microorganism using the above method.
The method is suitable for culturing photosynthetic microorganisms, and can obtain high-quality photosynthetic microorganism fermentation broth with higher yield under lower energy consumption.
The present invention will be described in detail by examples.
In the following examples:
determination of Chlorella dry weight: taking a proper amount of algae liquid, centrifuging for 5min at 6000r/min, removing supernatant, freeze-drying algae mud for 72h, and weighing.
Chlorella species were from the institute of aquatic organisms at the national academy of sciences. In the preparation stage of algae seed, adding about 600mL of BG11 culture medium and 5g/L glucose into a triangular flask, sterilizing at 120 ℃ for 30min, cooling, adding a proper amount of algae seed and 50mg/L kanamycin, and introducing sterile air at the light intensity of 6000lux and the temperature of 28 ℃ for culturing for about 3d to obtain the induced algae seed.
Composition of BG11 medium: k (K) 2 HPO 4 ·3H 2 O：40mg/L，NaNO 3 ：1500mg/L，Na 2 CO 3 ：20mg/L，MgSO 4 ·7H 2 O：75mg/L，CaCl 2 ·2H 2 O:36mg/L, citric acid: 6mg/L, ferric ammonium citrate: 6mg/L, sodium EDTA: 1mg/L, trace element A5:1ml/L.
Composition of trace element A5: h 3 BO 3 ：2860mg/L，MnCl 2 ·4H 2 O：1810mg/L，ZnSO 4 ·7H 2 O：222mg/L，CuSO 4 ·5H 2 O：79mg/L，NaMoO 4 ·5H 2 O：390mg/L，Co(NO 3 ) 2 ·6H 2 O：50mg/L。
Culture system: the culture system comprises a first culture unit and a second culture unit, wherein the first culture unit is a closed fermentation tank with the volume of 5L, and the second culture unit is a closed tubular type photobioreactor with the volume of 10L; wherein, the bottom algae liquid outlet of the closed fermentation tank is connected with an algae liquid conveying pipe, the algae liquid conveying pipe is connected to a conveying pump, and the outlet of the conveying pump is connected to an algae liquid inlet at the upper part of the closed tubular photobioreactor through another algae liquid conveying pipe; the top exhaust port of the closed fermentation tank is connected to the air inlet of the ventilation device in the closed tubular photo-bioreactor through the gas conveying pipe, the top exhaust port of the closed tubular photo-bioreactor is connected to the collecting tank, an air inlet is arranged at the joint of the collecting tank and the closed tubular photo-bioreactor, the gas exhaust port of the collecting tank is connected to the air compressor, and the exhaust port of the air compressor is connected to the air inlet of the ventilation device in the closed fermentation tank.
Example 1
This example is a description of the cultivation method of photosynthetic microorganisms of the present invention.
Adding 3L of BG11 culture medium and 15g/L glucose into a closed fermentation tank of the culture system, sterilizing at 120 ℃ for 30min, cooling for later use, adding glucose-induced chlorella algae and 50mg/L kanamycin during inoculation, introducing 1L/(L.min) of sterile air at 28 ℃ at the rotating speed of 250r/min, supplementing 15g/L glucose every day, supplementing other nutrient salts according to consumption, and culturing until algae cells grow slowly.
80% of algae liquid by volume from the closed fermentation tank is directly transferred into a closed tubular type photobioreactor without dilution for autotrophic culture, the culture temperature is 28 ℃, the illumination intensity is 3000lux, the illumination intensity is adjusted to 15000lux after 24 hours, and the illumination wavelength is 380-780nm. Meanwhile, 2.4L of sterilized BG11 culture medium, 15g/L of glucose and 50mg/L of kanamycin are continuously added into the closed fermentation tank, nutrition is supplemented every day, and the rest of algae liquid is used as algae seeds to continuously perform heterotrophic culture in the closed fermentation tank.
Introducing gas exhausted from an exhaust port of a closed fermentation tank into a bottom air inlet of a closed tubular photo-bioreactor, and ventilating the tubular photo-bioreactor, wherein the ventilation flow is kept at 1L/(L.min); and meanwhile, collecting and mixing the gas discharged from the exhaust port of the tubular photobioreactor with air, compressing the gas and delivering the mixture to the bottom air inlet of the closed fermentation tank, and ventilating the closed fermentation tank, wherein the ventilation flow is kept at 1L/(L.min).
The dry weight, the green a/dry weight (weight ratio) and the pH value of Chlorella in the fermenter and the tubular photobioreactor were measured at the designated time while the heterotrophic culture and the autotrophic culture were simultaneously carried out, and are shown in Table 1, table 2 and Table 3, respectively.
Example 2
This example is a description of the cultivation method of photosynthetic microorganisms of the present invention.
Adding 3L of BG11 culture medium and 15g/L glucose into a closed fermentation tank of the culture system, sterilizing at 120 ℃ for 30min, cooling for later use, adding glucose-induced chlorella species and 10mg/L chloramphenicol during inoculation, introducing 0.8L/(L.min) of sterile air, at 28 ℃, rotating at 250r/min, supplementing 15g/L glucose every day, supplementing other nutrient salts according to consumption, and culturing until algae cells grow slowly.
Transferring 80% by volume of algae liquid from a closed fermentation tank into a closed tubular photobioreactor without dilution for autotrophic culture, wherein the culture temperature is 28 ℃, the illumination intensity is 2000lux, the illumination intensity after 24 hours is adjusted to 12000lux, the illumination wavelength is 380-780nm, and the light-dark period is 12 hours: and 12h. Meanwhile, 2.4L of sterilized BG11 culture medium, 15g/L of glucose and 10mg/L of chloramphenicol are continuously added into the closed fermentation tank, nutrition is supplemented every day, and the rest of algae liquid is used as algae seeds to continuously perform heterotrophic culture in the closed fermentation tank.
Introducing gas exhausted from an exhaust port of a closed fermentation tank into a bottom air inlet of a closed tubular photo-bioreactor, and ventilating the tubular photo-bioreactor, wherein the ventilation flow is kept at 0.8L/(L.min); meanwhile, the gas discharged from the exhaust port of the tubular photo-bioreactor is collected and mixed with air, and then compressed and sent to the bottom air inlet of the closed fermentation tank, and the closed fermentation tank is aerated, and the aeration flow is kept at 0.8L/(L.min).
The dry weight, the green a/dry weight (weight ratio) and the pH value of Chlorella in the fermenter and the tubular photobioreactor were measured at the designated time while the heterotrophic culture and the autotrophic culture were simultaneously carried out, and are shown in Table 1, table 2 and Table 3, respectively.
Example 3
This example is a description of the cultivation method of photosynthetic microorganisms of the present invention.
According to the method of example 1, except that when the heterotrophic culture and the autotrophic culture are performed simultaneously, the autotrophic culture is irradiated with light at an irradiation intensity of 1000lux for 24 hours and then irradiated with light at 20000lux; the dry weight, the green a/dry weight (weight ratio) and the pH value of Chlorella in the fermenter and the tubular photobioreactor were measured at the designated time while the heterotrophic culture and the autotrophic culture were simultaneously carried out, and are shown in Table 1, table 2 and Table 3, respectively.
Example 4
This example is a description of the cultivation method of photosynthetic microorganisms of the present invention.
According to the method of example 1, except that when the heterotrophic culture and the autotrophic culture are simultaneously performed, the autotrophic culture is irradiated at an irradiation intensity of 5000lux for 24 hours and then at 8000 lux; the dry weight, the green a/dry weight (weight ratio) and the pH value of Chlorella in the fermenter and the tubular photobioreactor were measured at the designated time while the heterotrophic culture and the autotrophic culture were simultaneously carried out, and are shown in Table 1, table 2 and Table 3, respectively.
Example 5
This example is a description of the cultivation method of photosynthetic microorganisms of the present invention.
According to the method of example 1, except that when the heterotrophic culture and the autotrophic culture are performed simultaneously, the illumination intensity is not divided into two stages in the autotrophic culture, but the culture is continued under 20000lux illumination; the dry weight, the green a/dry weight (weight ratio) and the pH value of Chlorella in the fermenter and the tubular photobioreactor were measured at the designated time while the heterotrophic culture and the autotrophic culture were simultaneously carried out, and are shown in Table 1, table 2 and Table 3, respectively.
Example 6
This example is a description of the cultivation method of photosynthetic microorganisms of the present invention.
According to the method of example 1, except that when the heterotrophic culture and the autotrophic culture are performed simultaneously, the light wavelength of the autotrophic culture is the full wavelength; the dry weight, the green a/dry weight (weight ratio) and the pH value of Chlorella in the fermenter and the tubular photobioreactor were measured at the designated time while the heterotrophic culture and the autotrophic culture were simultaneously carried out, and are shown in Table 1, table 2 and Table 3, respectively.
Comparative example 1
According to the method of example 1, except that when the heterotrophic culture and the autotrophic culture are simultaneously performed, air is introduced into the tubular photobioreactor instead of the gas discharged from the fermenter; the dry weight, the green a/dry weight (weight ratio) and the pH value of Chlorella in the fermenter and the tubular photobioreactor were measured at the designated time while the heterotrophic culture and the autotrophic culture were simultaneously carried out, and are shown in Table 1, table 2 and Table 3, respectively.
Comparative example 2
According to the method of example 1, except that pure air is introduced into the fermenter without the gas discharged from the tubular photobioreactor when the heterotrophic culture and the autotrophic culture are performed simultaneously; the dry weight, the green a/dry weight (weight ratio) and the pH value of Chlorella in the fermenter and the tubular photobioreactor were measured at the designated time while the heterotrophic culture and the autotrophic culture were simultaneously carried out, and are shown in Table 1, table 2 and Table 3, respectively.
TABLE 1
TABLE 2
TABLE 3 Table 3
The change of the dry weight of the chlorella in the fermentation tank in the table 1 with time is plotted as a chlorella growth curve shown in fig. 1, and the change of the dry weight of the chlorella in the tubular reactor in the table 1 with time is plotted as a chlorella growth curve shown in fig. 2; chlorophyll a/dry weight values of chlorella in the fermenter of Table 2 over time were plotted as the graph shown in FIG. 3, and chlorophyll a/dry weight values of chlorella in the tube reactor of Table 2 over time were plotted as the graph shown in FIG. 4; the pH values of the chlorella in the fermenter of Table 3 over time were plotted as shown in FIG. 5, and the pH values of the chlorella in the tube reactor of Table 3 over time were plotted as shown in FIG. 6.
As can be seen from the results shown in the table and the graph, the method of the invention strengthens the two processes of autotrophic culture and heterotrophic culture of microalgae, and can realize CO generated by heterotrophy 2 Is fixed by in situ biology; meanwhile, the invention can also implement autotrophic culture of high-concentration microalgaeThereby remarkably improving the quality of heterotrophic microalgae biomass.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (11)
1. A method for heterotrophic-autotrophic continuous cultivation of a photosynthetic microorganism, the method comprising: under the first ventilation, photosynthetic microorganisms are sent to a first culture unit for heterotrophic culture, and a first culture solution is obtained; the photosynthetic microorganisms are sent to a second culture unit, and autotrophic culture is carried out under illumination and second ventilation to obtain a second culture solution; in the method, the gas discharged by the first culture unit is used as part or all of the second ventilated gas source, and the gas discharged by the second culture unit is used as part or all of the first ventilated gas source; the photosynthetic microorganisms in the two culture stages are the same; the ventilation volume of the second ventilation is 0.2-5L/(L.min);
the illumination comprises first-stage illumination and second-stage illumination, wherein the light intensity of the first-stage illumination is 2000-5000lux, and the light intensity of the second-stage illumination is 6000-20000lux; the illumination adopts an artificial light source, and the distance between the artificial light sources in the light direction is 2-300mm; the illumination duration of the first stage is 10-36h, and the rest cultivation time is illuminated in the second stage after the illumination of the first stage;
the first culture unit and the second culture unit are communicated in series; the culture solution after heterotrophic culture until the growth of algae cells is slow is partially sent to a second culture unit for autotrophic culture, and part of the first culture solution sent to the second culture unit is not diluted, so that high-concentration autotrophic culture is carried out in the second culture unit;
the photosynthetic microorganism is chlorella;
heterotrophic cultureThe culture medium adopted is composed of the following components: k (K) 2 HPO 4 ·3H 2 O：20-50mg/L，NaNO 3 ：1200-2000mg/L，Na 2 CO 3 ：10-30mg/L，MgSO 4 ·7H 2 O：50-90mg/L，CaCl 2 ·2H 2 O:30-50mg/L, citric acid: 1-10mg/L, ferric ammonium citrate: 1-10mg/L, EDTA sodium: 0.5-2mg/L, trace element A5:0.5-2ml/L.
2. The method of claim 1, wherein the first stage illumination has a light intensity of 3000-4000lux; the light intensity of the second-stage illumination is 10000-15000lux.
3. The method of claim 1, wherein the gas discharged from the second culturing unit is collected and compressed as a source of a first aeration.
4. A method according to claim 3, wherein the gas and air discharged by the second culturing unit are mixed as a source of a first aeration.
5. The method of claim 1, wherein the ventilation of the first ventilation is 0.2-5L/(L-min).
6. The method of any one of claims 1-5, wherein the heterotrophic culture is at a temperature of 20-35 ℃;
the temperature of the autotrophic culture is 20-35 ℃.
7. The method according to claim 1, wherein the system for continuous cultivation of photosynthetic microorganisms in heterotrophic-autotrophic serial connection comprises: a first cultivation unit for heterotrophic cultivation of photosynthetic microorganisms and a second cultivation unit for autotrophic cultivation of photosynthetic microorganisms, wherein the first cultivation unit comprises a first ventilation device, the second cultivation unit comprises a lighting device and a second ventilation device,
the exhaust port of the first culture unit is communicated with the second air-passing device of the second culture unit, so that the air discharged by the first culture unit is used as a part or all of air source of the second air-passing device;
the exhaust port of the second culture unit is communicated with the first ventilation device of the first culture unit, so that the gas discharged by the second culture unit is used as a gas source of part or all of the first ventilation device;
the culture solution outlet of the first culture unit is communicated with the algae solution inlet of the second culture unit, so that photosynthetic microorganisms directly enter the second culture unit for autotrophic culture after heterotrophic culture in the first culture unit;
the illumination device adopts an artificial light source, and the distance between the artificial light sources in the light direction is 2-300mm.
8. The method of claim 7, wherein the exhaust of the second culturing unit is connected to the gas collecting device and the pressurizing device in sequence, and then connected to the first ventilating device of the first culturing unit, so as to collect and compress the gas discharged from the second culturing unit as the gas source of the first ventilating device.
9. The method of claim 7 or 8, wherein the first culturing unit is a fermenter structure; the second culturing unit is in a tubular, plate-type or column-type photobioreactor structure.
10. A method of producing biomass comprising culturing photosynthetic microorganisms using the method of any one of claims 1-9 and extracting biomass from the resulting photosynthetic microorganisms.
11. A method of producing bioenergy comprising culturing a photosynthetic microorganism using the method of any one of claims 1-9.
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