Microalgae amplification culture device and culture method thereof

Abstract

The invention relates to the technical field of microbiology devices, in particular to a microalgae amplification culture device and a culture method.The microalgae amplification culture device comprises a culture box body and a cover plate, a reactor body used for culturing microalgae is fixedly installed in the culture box body, and a cleaning assembly used for cleaning the inner wall of the reactor body is arranged outside the reactor body; an adjusting assembly and a positioning assembly are arranged outside the reactor body, the interior of the reactor body is subjected to water supply cleaning through the cleaning assembly, meanwhile, the adjusting assembly is connected with the cleaning assembly in cooperation with the arranged positioning assembly, the adjusting assembly provides upward or downward power for an annular cleaning brush in the cleaning assembly, and the cleaning effect is improved. An electric driving stirring rod is matched to drive an annular cleaning brush to rotate in the reactor main body, and the annular cleaning brush is in rotating contact with the interior of the reactor main body and reciprocates up and down to thoroughly clean algal microorganisms in the reactor main body, so that the effect of ensuring a microalgae culture environment is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of microbiological devices, and in particular to a microalgae amplification culture device and a culture method thereof. Background Art

[0002] Microalgae have high plasticity and can synthesize a large number of high-value compounds under specific conditions. They have great potential in synthesizing large amounts of ALA and lutein. As a model microalgae for exploring lipid metabolism and photosynthesis mechanisms, Chlamydomonas reinhardtii cells proliferate very rapidly and can reach the logarithmic phase in about two days of culture. Its growth cycle is significantly shorter than that of most other microalgae. It has been widely used as a cell factory for the biosynthesis of some pigments, antibiotics, fatty acids and other active substances. It has broad application prospects in the fields of food, medicine, health food and biofuels. The optimal growth conditions and accumulation of high-value active substances of different microalgae are not the same. All-round regulation of nutrition and environmental factors is an effective measure to simultaneously increase Chlamydomonas reinhardtii ALA, lutein and biomass. Therefore, cultivation in a specific incubator is more conducive to controlling its stability in the optimal culture conditions.

[0003] Microalgae are cultured by setting up a reactor in an incubator. After the microalgae are cultured in the reactor and discharged, the algae-like microorganisms produced by the microalgae culture tend to adhere to the inner wall of the reactor. When the inside of the reactor is cleaned with clean water, it is not easy to completely clean the algae-like microorganisms from the inner wall of the reactor, which may lead to contamination of the microalgae when it is cultured again. Summary of the invention

[0004] In order to overcome the deficiencies of the prior art, the present invention provides a microalgae amplification culture device and a culture method thereof.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions: a microalgae amplification and cultivation device, comprising a culture box body and a cover plate, wherein one end of the culture box body close to the cover plate is set to an opening shape, an illumination lamp for providing illumination is fixedly inlaid on the inner wall of the cover plate, a temperature control device is arranged at the bottom of the culture box body, a temperature and humidity sensor is arranged inside the culture box body, a pH and cell counting sensor is arranged inside the reactor body, a control panel for controlling the internal equipment of the culture box body is fixedly connected to the outside of the cover plate, heat insulation plates are installed on both sides of the inner wall of the culture box body, the culture box body and the cover plate are connected by a rotating hinge, and the culture box body is connected to the cover plate by a rotating hinge. A reactor body for cultivating microalgae is fixedly installed inside the culture box body, an electrically driven stirring rod for stirring is arranged inside the reactor body, and the electrically driven stirring rod is driven by a stirring motor, the upper end of the reactor body is open for placing cultivated microalgae, a discharge pipe opening is fixedly connected to the outside of the reactor body, and the end of the discharge pipe opening away from the reactor body movably penetrates the culture box body and extends to the outside of the culture box body, a discharge valve is arranged outside the discharge pipe opening for controlling the switch, and a cleaning component for cleaning the inner wall of the reactor body is arranged outside the reactor body, and the cleaning component includes a rotating disk and a ring for cleaning the inner wall of the reactor body. The invention relates to a cleaning brush, wherein the rotating disk is movably connected to the outside of the electrically driven stirring rod, the outside of the rotating disk is provided with a rotating ring groove, the ring-shaped cleaning brush is fixedly connected to the outside of the rotating disk, and the cleaning component also includes a top sleeve and a sweeping plate for cleaning the ring-shaped cleaning brush, the outside of the top sleeve is fixedly connected with a bracket, one end of the bracket away from the top sleeve is fixedly connected to the outside of the reactor body, both sides of the outside of the top sleeve are provided with arc grooves connected to the inside of the top sleeve, the sweeping plate is fixedly connected to the inside of the top sleeve, both sides of the outside of the top sleeve are fixedly inlaid with connecting water pipes, the connecting water pipes are connected to the inside of the top sleeve, and one end of the connecting water pipes away from the top sleeve is fixed to penetrate the inner wall of the incubator body and to the incubator The main body extends outside and is connected to the external water pipe by a connecting water pipe to supply water to the top sleeve. The outside of the reactor body is provided with an adjustment component and a positioning component for controlling the rotating disk and the annular cleaning brush to move up and down inside the reactor body. The cleaning component also includes three vertical grooves. The three vertical grooves are evenly divided and opened on the outside of the electric-driven stirring rod. A short plate is slidably connected to the inside of each vertical groove. The end of the short plate away from the inside of the vertical groove is fixedly connected to the inner wall of the rotating disk. The rotating disk slides upward or downward on the outside of the electric-driven stirring rod through the vertical groove and the rotating disk. The inside of the rotating disk is provided with a slotted opening that matches the width of the position of the paddle blades on the outside of the electric-driven stirring rod.

[0006] As a preferred technical solution of the present invention, the positioning component includes a convex plate, which is movably connected to the inside of the rotating ring groove, and a slot for connection is provided on the outside of the convex plate. The positioning component also includes a card slot and two balls. The card slot is provided on the inner wall of the arc slot, and the two balls are rotatably embedded on both sides of the outside of the convex plate, and the two balls are respectively attached to both sides of the inner wall of the rotating ring groove. The outside of the convex plate is fixedly connected to a rotating base, and the inside of the rotating base is rotatably connected to a card plate, and the side of the card plate away from the rotating base is movably engaged with the inside of the card slot.

[0007] As a preferred technical solution of the present invention, the adjustment component includes a pull plate, which is arranged inside the incubator body, and the connecting plug plate is fixedly connected to the outside of the pull plate. The shape and size of the connecting plug plate are adapted to the slot. The adjustment component also includes two support plates and rotating rollers. The two support plates are fixedly connected to the inner wall of the incubator body, and the two rotating rollers are respectively rotatably connected to the outside of the two support plates. The rotating roller on one side is fixedly connected to a motor motor on the outside, and the output shaft of the motor motor movably penetrates the support plate and is fixedly connected to the rotating roller. The outsides of the two side plates are movably sleeved with a transmission belt, and the outside of the transmission belt is fixedly connected to the side plate. The side plate is fixedly connected to the long plate at one end away from the transmission belt, and the long plate is movably connected to the cross plate inside the long plate. The end of the cross plate away from the long plate is fixedly connected to the pull plate. The adjustment component also includes a limiting slot, a side beam plate and a limiting plate. The limiting slot is opened on the outside of the long plate, the side beam plate is fixedly connected to the inside of the incubator body, the limiting plate is fixedly connected to one end of the side beam plate close to the long plate, and the side beam plate and the limiting plate are movably connected inside the limiting slot.

[0008] A cultivation method of a microalgae amplification cultivation device comprises the following steps:

[0009] S1: fresh Chlamydomonas reinhardtii cells cultured to the rapid division phase are inoculated into a nutrient solution containing 0-2.5 g tris(hydroxymethyl)aminomethane, 4-18 g glucose monohydrate, 2-6 mg 6-benzyladenine, 2-6 mg 1-naphthaleneacetic acid, 0-10 g Porphyra extract and 1 portion of TAP medium without Tris per liter at a dry biomass of 0.15-0.25 g / L;

[0010] S2: then adding the mixed solution into the reactor body in the incubator body for culturing;

[0011] S3: Set the temperature in the incubator, light intensity, and stirring speed in the reactor to 9.5-10.5°C, 40-125 μE / m 2 / s and 400-2000rpm, continuous light culture for 120-170h, achieve a significant increase in the simultaneous production of α-linolenic acid and lutein in Chlamydomonas reinhardtii, and the yields of both are significantly higher than those under conventional culture conditions;

[0012] S4: After the above mixed liquid culture is completed, the algae liquid is discharged from the discharge pipe outlet below the reactor body. At this time, the inside of the reactor body is quickly cleaned with the cleaning component and the regulating component.

[0013] As a preferred technical solution of the present invention, the preparation process of laver extract is to chop fresh laver in the middle and late stages of maturity into pieces with a size of less than 0.5 mm, mix it with an appropriate amount of pure water, and ultrasonically crush it for 30 minutes, and obtain it after sterilization. The mass volume ratio of fresh laver to pure water is 1:20-50.

[0014] Compared with the prior art, the present invention can achieve the following beneficial effects:

[0015] 1. After amplifying and culturing the microalgae inside the reactor body, the cultured microalgae liquid is discharged from the discharge pipe port, and the cleaning component is used to send water to clean the inside of the reactor body. At the same time, the positioning component is used to connect the adjustment component with the cleaning component. The adjustment component provides upward or downward power for the annular cleaning brush in the cleaning component, and cooperates with the electric-driven stirring rod to drive the annular cleaning brush to rotate inside the reactor body. The annular cleaning brush is in contact with the rotation inside the reactor body and performs up and down reciprocating motion to thoroughly clean the algae-like microorganisms inside the reactor body, thereby achieving the effect of ensuring the microalgae cultivation environment.

[0016] 2. After the card plate is rotated with the inside of the rotating base as the axis and enters the inside of the card slot, the rotating disk is fixed inside the top sleeve by the friction force between the card plate and the inside of the card slot. At this time, the electric drive stirring rod is rotated to drive the rotating disk and the annular cleaning brush to rotate inside the top sleeve. The annular cleaning brush rotates and contacts the sweeping plate, and the sweeping plate strikes and cleans the annular cleaning brush. By quickly cleaning the annular cleaning brush, secondary pollution of the inner wall of the reactor body caused by the annular cleaning brush when it cleans the inner wall of the reactor body reciprocatingly is avoided, thereby further improving the cleaning effect of the inside of the reactor body.

[0017] 3. After the connecting plug plate is inserted into the slot and connected to the convex plate, when the electric-driven stirring rod drives the rotating disk to rotate, the convex plate drives the rolling ball to rotate inside the rotating ring groove, and the rolling ball positions the convex plate and assists the rotating disk to move outside the convex plate, thereby ensuring the stable rotation of the rotating disk outside the electric-driven stirring rod, and at the same time does not affect the downward squeezing of the rotating disk by the convex plate, so that the rotating disk drives the annular cleaning brush to push upward or downward for cleaning while performing stable rotation cleaning.

[0018] 4. The method of the present invention can significantly promote the growth of Chlamydomonas reinhardtii, the biosynthesis of α-linolenic acid and lutein, and increase its biomass, α-linolenic acid and lutein content by combining nutrition with environmental factors in an all-round way. The above method can significantly increase its soluble sugar content per unit dry weight, and can effectively achieve multi-product co-production, which is of great value for further promoting the industrial application of Chlamydomonas reinhardtii. The device of the present invention is simple to operate, and can accurately control the low temperature conditions required for Chlamydomonas reinhardtii to simultaneously and efficiently synthesize α-linolenic acid and lutein, while meeting the requirements of algae liquid stirring and rapid cleaning of the reactor body, effectively simplifying the number of equipment required for microalgae cultivation, significantly improving efficiency, shortening time and reducing costs, and can more quickly and efficiently scale up the cultivation of high-value microalgae without seasonal restrictions. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the response surface analysis of the effects of photoperiod, glucose, 6-benzyladenine and 1-naphthaleneacetic acid on the biomass of Chlamydomonas reinhardtii in the present invention;

[0020] Figure 2 This is a schematic diagram of the response surface analysis of the effects of light cycle, glucose, 6-benzyladenine and 1-naphthaleneacetic acid on the α-linolenic acid content of Chlamydomonas reinhardtii in the present invention;

[0021] Figure 3 This is a schematic diagram of the response surface analysis of the effects of light cycle, glucose, 6-benzyladenine and 1-naphthaleneacetic acid on the yield of α-linolenic acid in Chlamydomonas reinhardtii;

[0022] Figure 4 It is a bar graph of the biomass accumulation of Chlamydomonas reinhardtii under different culture conditions of the present invention;

[0023] Figure 5 The biomass yield change bar graph of Chlamydomonas reinhardtii under different culture conditions of the present invention;

[0024] Figure 6 The bar graph is a graph showing the accumulation of α-linolenic acid, lutein and soluble sugar in Chlamydomonas reinhardtii under different culture conditions of the present invention;

[0025] Figure 7 It is a structural schematic diagram of the main body of the incubator of the present invention;

[0026] Figure 8 It is a structural schematic diagram of the cover plate of the present invention;

[0027] Fig. 9 It is a partial structural schematic diagram of the incubator body of the present invention;

[0028] Fig.10 It is a partial cross-sectional structural schematic diagram of the incubator body of the present invention;

[0029] Fig.11 It is a structural schematic diagram of the reactor body of the present invention;

[0030] Fig.12 It is a structural schematic diagram of the rotating disk of the present invention;

[0031] Fig.13 It is a structural schematic diagram of the convex plate of the present invention;

[0032] Fig.14 It is a schematic diagram of the structure of the side beam plate of the present invention;

[0033] Fig.15 It is a schematic structural diagram of the top sleeve of the present invention.

[0034] Among them: 10, incubator body; 11, cover plate; 12, rotating hinge; 13, control panel; 14, lighting tube; 15, reactor body; 16, discharge pipe; 17, electric drive stirring rod; 20, top sleeve; 21, arc notch; 22, bracket; 23, connecting water pipe; 24, sweeping plate; 25, vertical groove; 26, rotating disk; 27, annular cleaning brush; 28, short plate; 29, rotating ring notch; 30, support plate; 31, rotating roller; 32, transmission belt; 33, motor; 34, side plate; 35, long plate; 36, limit notch; 37, side beam plate; 38, pull plate; 39, connecting plug plate; 310, horizontal plate; 311, limit plate; 40, convex plate; 41, card plate; 42, rotating base; 43, slot; 44, rolling ball; 45, card notch. DETAILED DESCRIPTION

[0035] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. The experimental methods in the following embodiments, unless otherwise specified, are conventional methods, and the materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0036] Embodiment 1

[0037] like Figure 7-Figure 15The microalgae amplification culture device shown in the figure comprises a culture box body 10 and a cover plate 11, wherein one end of the culture box body 10 close to the cover plate 11 is set to be open, and the inner wall of the cover plate 11 is fixedly inlaid with a light tube 14 for providing light, and the bottom of the culture box body 10 is provided with a temperature control device, the interior of the culture box body 10 is provided with a temperature and humidity sensor, and the interior of the reactor body 15 is provided with a pH and cell count sensor, and the outside of the cover plate 11 is fixedly connected with a control panel 13 for controlling the internal equipment of the culture box body 10, and both sides of the inner wall of the culture box body 10 are installed with heat insulation boards, and the culture box body 10 and the cover plate 11 are connected by a rotating hinge 12, and the interior of the culture box body 10 is fixedly installed with a reactor for culturing microalgae. The reactor body 15 is provided with an electrically driven stirring rod 17 for stirring inside the reactor body 15, and the electrically driven stirring rod 17 is driven by a stirring motor. The upper end of the reactor body 15 is open for placing cultured microalgae. The outside of the reactor body 15 is also provided with a sterilizing device for sterilizing bacteria remaining after cleaning. The outside of the reactor body 15 is fixedly connected with a drain pipe 16. The end of the drain pipe 16 away from the reactor body 15 is movable to penetrate the incubator body 10 and extend to the outside of the incubator body 10. The outside of the drain pipe 16 is provided with a drain valve for controlling the switch. The outside of the reactor body 15 is provided with a cleaning component for cleaning the inner wall thereof. The cleaning component includes a cleaning device for cleaning the reactor body 1 5, the rotating disk 26 and the annular cleaning brush 27 are movably connected to the outside of the electrically driven stirring rod 17, the rotating disk 26 is provided with a rotating ring notch 29 on the outside, the annular cleaning brush 27 is fixedly connected to the outside of the rotating disk 26, the cleaning assembly further comprises a top sleeve 20 and a sweeping plate 24 for cleaning the annular cleaning brush 27, the outside of the top sleeve 20 is fixedly connected with a bracket 22, one end of the bracket 22 away from the top sleeve 20 is fixedly connected to the outside of the reactor body 15, both sides of the outside of the top sleeve 20 are provided with arc notches 21 communicating with the inside thereof, the sweeping plate 24 is fixedly connected to the inside of the top sleeve 20, both sides of the outside of the top sleeve 20 are fixedly inlaid with connecting water pipes 23, the connecting water pipes 23 are connected with the inside of the top sleeve 20, and the connecting water pipes 23 are connected with the inside of the top sleeve 20, and the connecting water pipes 23 are connected with the inside of the top sleeve 20. The end away from the top cover 20 is fixedly penetrated through the inner wall of the incubator body 10 and extends to the outside of the incubator body 10, and the connecting water pipe 23 is connected to the external water pipe to supply water to the inside of the top cover 20. The outside of the reactor body 15 is provided with an adjustment component and a positioning component for controlling the rotating disk 26 and the annular cleaning brush 27 to move up and down inside the reactor body 15. The cleaning component also includes three vertical grooves 25, which are evenly divided and opened on the outside of the electric-driven stirring rod 17. A short plate 28 is slidably connected to the inside of each vertical groove 25. The end of the short plate 28 away from the inside of the vertical groove 25 is fixedly connected to the inner wall of the rotating disk 26. The rotating disk 26 slides upward or downward on the outside of the electric-driven stirring rod 17 through the vertical groove 25 and the rotating disk 26.The inside of the rotating disk 26 is provided with a slotted opening that matches the width of the outer blades of the electrically driven stirring rod 17.

[0038] The positioning assembly includes a convex plate 40, which is movably connected to the inside of the rotating ring groove 29, and a slot 43 for connection is provided on the outside of the convex plate 40. The positioning assembly also includes a card slot 45 and two rolling balls 44. The card slot 45 is provided on the inner wall of the arc slot 21, and the two rolling balls 44 are respectively rotatably embedded on both sides of the outside of the convex plate 40, and the two rolling balls 44 are respectively attached to both sides of the inner wall of the rotating ring groove 29, and the outside of the convex plate 40 is fixedly connected to a rotating base 42, and the inside of the rotating base 42 is rotatably connected to a card plate 41, and the side of the card plate 41 away from the rotating base 42 is movably engaged with the inside of the card slot 45, and the card plate 41 is rotated to rotate the base 42. After the inner part of the top sleeve 20 is rotated by the axis and enters the inner part of the card slot 45, the friction force between the card plate 41 and the inner part of the card slot 45 fixes the rotating disc 26 inside the top sleeve 20. At this time, the electrically driven stirring rod 17 is rotated to drive the rotating disc 26 and the annular cleaning brush 27 to rotate inside the top sleeve 20. The annular cleaning brush 27 rotates and contacts with the sweeping plate 24, and the sweeping plate 24 strikes and cleans the annular cleaning brush 27. By quickly cleaning the annular cleaning brush 27, secondary pollution of the inner wall of the reactor body 15 caused by the annular cleaning brush 27 when cleaning the inner wall of the reactor body 15 back and forth is avoided, and the cleaning effect of the inside of the reactor body 15 is further improved.

[0039] The adjusting component includes a pull plate 38, which is arranged inside the incubator body 10, a connecting plug plate 39 fixedly connected to the outside of the pull plate 38, and the connecting plug plate 39 is adapted to the shape and size of the slot 43. The adjusting component also includes two support plates 30 and a rotating roller 31. The two support plates 30 are fixedly connected to the inner wall of the incubator body 10, and the two rotating rollers 31 are rotatably connected to the outside of the two support plates 30 respectively. The outside of the rotating roller 31 on one side is fixedly connected to a motor 33, and the output shaft of the motor 33 movably penetrates the support plate 30 and is fixedly connected to the rotating roller 31. The outside of the two side plates 34 is movably sleeved with a transmission belt 32, and the outside of the transmission belt 32 is fixedly connected to the side plate 34, and the side plate 34 is fixedly connected to the end of the side plate 34 away from the transmission belt 32. The long plate 35 is internally movably connected to a transverse plate 310, and the end of the transverse plate 310 away from the long plate 35 is fixedly connected to the pull plate 38. The adjusting component also includes a limited position The slot 36, the side beam 37 and the limiting plate 311, the limiting slot 36 is arranged outside the long plate 35, the side beam 37 is fixedly connected to the inside of the incubator body 10, the limiting plate 311 is fixedly connected to one end of the side beam 37 close to the long plate 35, the side beam 37 and the limiting plate 311 are movably connected inside the limiting slot 36, after the connecting plug plate 39 is inserted into the slot 43 and connected with the convex plate 40, when the electric drive stirring rod 17 drives the rotating disk 26 to rotate, the convex plate 40 drives the rolling ball 44 to rotate inside the rotating ring slot 29, the rolling ball 44 positions the convex plate 40 and assists the rotating disk 26 to move outside the convex plate 40, thereby ensuring the stable rotation of the rotating disk 26 outside the electric drive stirring rod 17, and at the same time does not affect the convex plate 40 pushing the rotating disk 26 downward, so that the rotating disk 26 drives the annular cleaning brush 27 to push upward or downward for cleaning while performing stable rotation cleaning.

[0040] After the microalgae are enlarged and cultured inside the reactor body 15, the cultured microalgae liquid is discharged from the discharge pipe port 16. At this time, water is introduced into the reactor body 15 through the connecting water pipe 23 to clean the algae-like microorganisms remaining inside the reactor body 15. At the same time, the cover plate 11 is rotated outwardly with the rotating hinge 12 as the center to open the inside of the incubator body 10. The output shaft of the motor 33 drives the rotating roller 31 to rotate the transmission belt 32. The transmission belt 32 drives the side plate 34 to move the long plate 35 upward inside the incubator body 10. The long plate 35 drives the pull plate 38 upward and moves the connecting plug plate 39 to the slot 43. After the pull plate 38 is in position, the cross plate 310 is driven by the pull plate 38 to move the connecting plug plate 39 laterally outside the long plate 35, and the pull plate 38 drives the connecting plug plate 39 to engage into the inside of the slot 43. At this time, the pull plate 38 is connected to the convex plate 40, and the connecting plug plate 39 drives the convex plate 40 downward to push the rotating disk 26 downward, and the rotating disk 26 drives the annular cleaning brush 27 to clean the inner wall of the reactor body 15. At the same time, the electrically driven stirring rod 17 is rotated to drive the short plate 28 so that the rotating disk 26 drives the annular cleaning brush 27 to rotate and contact the inner wall of the reactor body 15, thereby improving the cleaning effect of the algae-like microorganisms remaining on the inner wall of the reactor body 15.

[0041] A cultivation method of a microalgae amplification cultivation device comprises the following steps:

[0042] S1: fresh Chlamydomonas reinhardtii cells cultured to the rapid division stage are inoculated at a dry biomass of 0.15-0.25 g / L into a nutrient solution containing 0-2.5 g tris(hydroxymethyl)aminomethane, 4-18 g glucose monohydrate, 2-6 mg 6-benzyladenine, 2-6 mg 1-naphthaleneacetic acid, 0-10 g laver extract and 1 portion of TAP medium without Tris per liter, wherein the preparation process of the laver extract is to chop fresh laver in the middle and late stages of maturity into pieces with a size of less than 0.5 mm, mix with an appropriate amount of pure water, and ultrasonically crush for 30 minutes, and obtain after sterilization, wherein the mass volume ratio of fresh laver to pure water is 1:20-50;

[0043] S2: then adding the mixed solution into the reactor body 15 in the incubator body 10 for incubation;

[0044] S3: The temperature and light intensity in the incubator body 10 and the stirring speed in the reactor body 15 are set to 9.5-10.5°C, 40-125 μE / m2 / s and 400-2000 rpm, respectively, and the continuous illumination culture is carried out for 120-170 hours, so that the production of α-linolenic acid and lutein in Chlamydomonas reinhardtii is significantly improved, and the yields of both are significantly higher than those under conventional culture conditions;

[0045] S4: After the above-mentioned mixed liquid culture is completed, the algae liquid is discharged from the discharge pipe 16 below the reactor body 15. At this time, the cleaning component and the adjustment component are used to quickly clean the inside of the reactor body 15.

[0046] like Figure 1-3 As shown in the figure, with photoperiod, glucose, 6-benzyladenine and 1-naphthaleneacetic acid as variables, and biomass, α-linolenic acid content and yield as responses, the response surface methodology was used to inoculate fresh Chlamydomonas reinhardtii cells cultured to the rapid division stage at a dry biomass of 0.18 g / L into a nutrient solution containing 2.42 g tris(hydroxymethylaminomethane), 4-8 g glucose, 2-6 mg6-benzyladenine, 2-6 mg1-naphthaleneacetic acid, 0.01 g laver extract and 1 portion of TAP medium without Tris per liter, and cultured at 10°C, 50 μE / m2 / s, and 8 / 24-24 / 0h day / night ratio for 144 h to predict and verify the optimal levels of key factors.

[0047] in accordance with Figure 1 As shown in Table 1, the photoperiod, glucose and 6-benzyladenine all had a significant effect on the biomass of Chlamydomonas reinhardtii, but 1-naphthaleneacetic acid had no significant effect on the biomass of Chlamydomonas reinhardtii. Figure 2 As shown in Table 2, among the four key factors, the photoperiod has a very significant effect on the α-linolenic acid content of Chlamydomonas reinhardtii. The effects of glucose, 6-benzyladenine and 1-naphthaleneacetic acid alone on the α-linolenic acid content of Chlamydomonas reinhardtii are not significant, but the combined effects of photoperiod, 6-benzyladenine and 1-naphthaleneacetic acid have a significant effect on the α-linolenic acid content of Chlamydomonas reinhardtii. Figure 3 As shown in Table 3, the three factors of photoperiod, glucose and 1-naphthylacetic acid alone have a very significant effect on the α-linolenic acid yield of Chlamydomonas reinhardtii, and the combined effect of photoperiod and glucose has a significant effect on the α-linolenic acid yield of Chlamydomonas reinhardtii, but 6-benzyladenine has no significant effect on the α-linolenic acid yield.

[0048] Table 1: ANOVA table of biomass regression equation:

[0049] Sources of variance sum of squares Degrees of Freedom Mean Square F-number P-value Model 0.5347 14 0.0382 120.57 <0.0001 A-PP 0.4126 1 0.4126 1302.34 <0.0001 B-Glu 0.0408 1 0.0408 128.9 <0.0001 C-BAP 0.0057 1 0.0057 18.13 0.0008 D-NAA 0.0011 1 0.0011 3.33 0.0895 AB 0.0002 1 0.0002 0.4932 0.494 AC 0.0004 1 0.0004 1.11 0.31 AD 0.0004 1 0.0004 1.11 0.31 BC 0 1 0 0 1 BD 0 1 0 0 1 CD 0.0006 1 0.0006 1.97 0.1819 <![CDATA[A 2 ]]> 0.0625 1 0.0625 197.16 <0.0001 <![CDATA[B 2 ]]> 0.002 1 0.002 6.27 0.0253 <![CDATA[C 2 ]]> 0.0001 1 0.0001 0.242 0.6304 <![CDATA[D 2 ]]> 0.0005 1 0.0005 1.68 0.2157 Residual 0.0044 14 0.0003 Lack of Fit 0.0024 10 0.0002 0.487 0.8376 Pure Error 0.002 4 0.0005 sum 0.5391 28 <![CDATA[R 2 =0.9918]]> <![CDATA[R 2 Adj=0.9835]]> CV=1.11%

[0050] Table 2: ANOVA table of ALA content regression equation:

[0051] Sources of variance sum of squares Degrees of Freedom Mean Square F-number P-value Model 185.39 14 13.24 107.4 <0.0001 A-PP 163.2 1 163.2 1323.64 <0.0001 B-Glu 0.0529 1 0.0529 0.4289 0.5232 C-BAP 0.1572 1 0.1572 1.27 0.2778 D-NAA 0.3665 1 0.3665 2.97 0.1067 AB 0.1866 1 0.1866 1.51 0.2388 AC 0.885 1 0.885 7.18 0.018 AD 1.43 1 1.43 11.6 0.0043 BC 0.0887 1 0.0887 0.7195 0.4106 BD 0.00 1 0.00 0.00 1 CD 0.0222 1 0.0222 0.18 0.6778 <![CDATA[A 2 ]]> 17.74 1 17.74 143.85 <0.0001 <![CDATA[B 2 ]]> 0.1419 1 0.1419 1.15 0.3015 <![CDATA[C 2 ]]> 0.058 1 0.058 0.4705 0.504 <![CDATA[D 2 ]]> 0.1023 1 0.1023 0.8299 0.3777 Residual 1.73 14 0.1233 Lack of Fit 1.4 10 0.1399 1.71 0.3189 Pure Error 0.3271 4 0.0818 sum 187.12 28 <![CDATA[R 2 =0.9908]]> <![CDATA[R 2 Adj=0.9816]]> CV=0.9634%

[0052] Table 3: ANOVA table of ALA yield regression equation:

[0053] Sources of variance sum of squares Degrees of Freedom Mean Square F-number P-value Model 963.38 14 68.81 303.53 <0.0001 A-PP 896.83 1 896.83 3955.89 <0.0001 B-Glu 11.59 1 11.59 51.12 <0.0001 C-BAP 0.2878 1 0.2878 1.27 0.2788 D-NAA 2.68 1 2.68 11.83 0.004 AB 1.47 1 1.47 6.47 0.0234 AC 0.0655 1 0.0655 0.2887 0.5995 AD 0.8279 1 0.8279 3.65 0.0767 BC 0.4292 1 0.4292 1.89 0.1905 BD 0.0019 1 0.0019 0.0083 0.9288 CD 0.083 1 0.083 0.3659 0.5549 <![CDATA[A 2 ]]> 43.57 1 43.57 192.17 <0.0001 <![CDATA[B 2 ]]> 0.0648 1 0.0648 0.2858 0.6013 <![CDATA[C 2 ]]> 0.5164 1 0.5164 2.28 0.1535 <![CDATA[D 2 ]]> 0.9043 1 0.9043 3.99 0.0656 Residual 3.17 14 0.2267 Lack of Fit 2.36 10 0.2363 1.16 0.4793 Pure Error 0.8112 4 0.2028 sum 966.56 28 <![CDATA[R 2 =0.9967]]> <![CDATA[R 2 Adj=0.9934]]> CV=1.7%

[0054] The results of the validation experiment showed that under the conditions of 8 g / L glucose, 2 mg / L BAP, 4.37 mg / L NAA and 24:0 h photoperiod, the biomass, α-linolenic acid content and α-linolenic acid yield of Chlamydomonas reinhardtii could reach 1.80 g / L, 40.69% and 39.19 mg / L / d, respectively, which were significantly higher than those under the conventional conditions (25°C, 50 μE / m 2 / s, 14 / 10h) culture was 95.65%, 39.35% and 2.41 times higher.

[0055] Embodiment 2

[0056] Based on the first embodiment, refer to Figure 1-6 After obtaining the best culture method, amplification verification was carried out. Fresh Chlamydomonas reinhardtii cells cultured to the rapid division stage were inoculated with a dry biomass of 0.18 g / L into a nutrient solution containing 2.42 g tris(hydroxymethyl)aminomethane), 8.71 g glucose monohydrate, 2.1 mg 6-benzyladenine, 4.3 mg 1-naphthaleneacetic acid, 0.01 g laver extract and 1 portion of TAP culture medium without tris per liter. 3 L of the above-mentioned mixed solution was added to the pre-sterilized incubator body 10. The temperature, light intensity and stirring speed of the incubator body 10 were set to 10°C, 50 μE / m 2 / s and 550rpm, and continuously illuminate for 144 hours. During the culture period, the pH value and cell count sensor dynamically record the change pattern of the pH value and cell number of the algae liquid in the reactor body 15. After the culture is completed, the algae liquid is discharged from the drain pipe 16 at the bottom of the reactor body 15. The Chlamydomonas reinhardtii culture liquid obtained by the above method is further dehydrated and dried for analysis and determination of active substances such as α-linolenic acid and lutein. The method can significantly increase the content of compounds such as α-linolenic acid and lutein in Chlamydomonas reinhardtii.

[0057] like Figure 4 The biomass accumulation of Chlamydomonas reinhardtii after culturing under conventional conditions and using the method of the present invention is compared. By comparing the dynamic changes in the biomass of Chlamydomonas reinhardtii under optimal and conventional conditions, it is found that with the extension of the culture time, the biomass of Chlamydomonas reinhardtii treated with different conditions shows a significant increasing trend, but the biomass under conventional conditions throughout the growth cycle is significantly lower than that under the above-mentioned implementation conditions. After 6 days of culture, the biomass of Chlamydomonas reinhardtii cultured using this method can reach 1.90 g / L.

[0058] like Figure 5As described above, the biomass yield of Chlamydomonas reinhardtii cultured under conventional conditions and by the method of the present invention is compared. Contrary to the changing trend of the biomass, with the increase of the culture time, the biomass yield under both conditions shows a decreasing trend. Except for 1-2 days of culture, the biomass yield of 3-6 days of culture under the optimal conditions is significantly higher than that of culture under conventional conditions. After 6 days of culture, the biomass yield of cultured under the above method can still reach 283.89 mg / L / d, which is significantly higher than 144.86 mg / L / d under conventional culture conditions. The changes in the content and yield of α-linolenic acid, lutein, and soluble sugars in Chlamydomonas reinhardtii cultured under conventional conditions and by the method of the present invention are as follows: Figure 6 As shown, after 6 days of cultivation, the content and yield of α-linolenic acid, lutein and soluble sugar of Chlamydomonas reinhardtii cultured by the above method were significantly higher than those under conventional conditions. The contents of the three indicators could reach 40.60%, 6.40 mg / g and 25.81%, respectively, which were 39.23%, 1.64 times and 1.72 times higher than those under conventional conditions, respectively. The yields of the three indicators could reach 40.81, 1.82 and 69.69 mg / L / d, respectively, which were 2.55, 2.94 and 2.85 times higher than those under conventional conditions, respectively.

[0059] Comprehensive analysis of the above results shows that the all-round regulation of nutrition combined with environmental factors can significantly promote the growth of Chlamydomonas reinhardtii, the biosynthesis of α-linolenic acid and lutein, and increase its biomass, α-linolenic acid and lutein content. The above method can significantly increase its soluble sugar content per unit dry weight, and can effectively achieve multi-product co-production, which is of great value for further promoting the industrial application of Chlamydomonas reinhardtii. The device of the present invention is simple to operate, and can accurately control the low temperature conditions required for Chlamydomonas reinhardtii to simultaneously and efficiently synthesize α-linolenic acid and lutein, while meeting the requirements of algae liquid stirring and rapid cleaning of the reactor body 15, effectively simplifying the number of equipment required for microalgae cultivation, significantly improving efficiency, shortening time and reducing costs, and can more quickly and efficiently scale up the cultivation of high-value microalgae without seasonal restrictions.

[0060] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited thereto, and various changes can be made within the knowledge scope of technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A microalgae amplification culture device, comprising a culture box body (10) and a cover plate (11), wherein one end of the culture box body (10) close to the cover plate (11) is set to be open, and the inner wall of the cover plate (11) is fixedly inlaid with a lighting tube (14) for providing light, and the bottom of the culture box body (10) is provided with a temperature control device, and the outside of the cover plate (11) is fixedly connected with a control panel (13) for controlling the internal equipment of the culture box body (10), and both sides of the inner wall of the culture box body (10) are installed with heat insulation boards, and the culture box body (10) and the cover plate (11) are connected by a rotating hinge (12), characterized in that: A reactor body (15) for culturing microalgae is fixedly installed inside the culture box body (10); an electrically driven stirring rod (17) for stirring is arranged inside the reactor body (15); the electrically driven stirring rod (17) is driven by a stirring motor; a liquid discharge nozzle (16) is fixedly connected to the outside of the reactor body (15); and a cleaning component for cleaning the inner wall of the reactor body (15) is arranged outside the reactor body (15); The cleaning assembly comprises a rotating disk (26) and an annular cleaning brush (27) for cleaning the inner wall of the reactor body (15); the rotating disk (26) is movably connected to the outside of the electrically driven stirring rod (17); a rotating ring notch (29) is provided on the outside of the rotating disk (26); and the annular cleaning brush (27) is fixedly connected to the outside of the rotating disk (26); The cleaning assembly also includes a top sleeve (20) and a sweeping plate (24) for cleaning the annular sweeping brush (27); the outside of the top sleeve (20) is fixedly connected to a bracket (22); one end of the bracket (22) away from the top sleeve (20) is fixedly connected to the outside of the reactor body (15); both sides of the outside of the top sleeve (20) are provided with arc notches (21) communicating with the inside of the top sleeve (20); the sweeping plate (24) is fixedly connected to the inside of the top sleeve (20); both sides of the outside of the top sleeve (20) are fixedly inlaid with connecting water pipes (23); the connecting water pipes (23) are connected to the inside of the top sleeve (20); and one end of the connecting water pipes (23) away from the top sleeve (20) is fixedly penetrated through the inner wall of the incubator body (10) and extends to the outside of the incubator body (10); The outside of the reactor body (15) is provided with an adjustment component and a positioning component for controlling the rotating disk (26) and the annular cleaning brush (27) to move up and down inside the reactor body (15). The positioning component includes a convex plate (40), which is movably connected to the inside of the rotating ring groove (29). The outside of the convex plate (40) is provided with a slot (43) for connection. The adjustment component includes a pull plate (38), which is arranged inside the incubator body (10). The connecting plug plate (39) is fixedly connected to the outside of the pull plate (38), and the shape and size of the connecting plug plate (39) are adapted to the slot (43).

2. A microalgae amplification culture device according to claim 1, characterized in that: The incubator body (10) is provided with temperature and humidity sensors inside, and the reactor body (15) is provided with pH and cell count sensors inside.

3. The microalgae amplification culture device according to claim 1, characterized in that: The cleaning assembly further comprises three vertical grooves (25), which are evenly spaced and arranged outside the electrically driven stirring rod (17), and a short plate (28) is slidably connected inside each vertical groove (25), and one end of the short plate (28) away from the inside of the vertical groove (25) is fixedly connected to the inner wall of the rotating disk (26).

4. The microalgae amplification culture device according to claim 1, characterized in that: The positioning assembly also includes a slot (45) and two rolling balls (44). The slot (45) is provided on the inner wall of the arc slot (21). The two rolling balls (44) are respectively rotatably embedded on the two sides of the outer side of the convex plate (40), and the two rolling balls (44) are respectively attached to the two sides of the inner wall of the rotating ring slot (29). The outer side of the convex plate (40) is fixedly connected to a rotating base (42), and the inner side of the rotating base (42) is rotatably connected to a clamping plate (41). The side of the clamping plate (41) away from the rotating base (42) is movably clamped in the inner side of the slot (45).

5. The microalgae amplification culture device according to claim 1, characterized in that: The adjustment component also includes two support plates (30) and a rotating roller (31). The two support plates (30) are fixedly connected to the inner wall of the incubator body (10). The two rotating rollers (31) are rotatably connected to the outside of the two support plates (30). The outside of the rotating roller (31) on one side is fixedly connected to a motor (33). The output shaft of the motor (33) movably penetrates the support plate (30) and is fixedly connected to the rotating roller (31). The outside of the two side plates (34) is movably sleeved with a transmission belt (32). The outside of the transmission belt (32) is fixedly connected to the side plate (34). The end of the side plate (34) away from the transmission belt (32) is fixedly connected to a long plate (35). The inside of the long plate (35) is movably connected to a transverse plate (310). The end of the transverse plate (310) away from the long plate (35) is fixedly connected to a pull plate (38).

6. The microalgae amplification culture device according to claim 5, characterized in that: The adjustment component further comprises a limiting slot (36), a side beam plate (37) and a limiting plate (311); the limiting slot (36) is arranged outside the long plate (35); the side beam plate (37) is fixedly connected to the inside of the incubator body (10); the limiting plate (311) is fixedly connected to one end of the side beam plate (37) close to the long plate (35); and the side beam plate (37) and the limiting plate (311) are movably connected inside the limiting slot (36).

7. The cultivation method of the microalgae enlargement cultivation device according to claim 1, characterized in that: The steps include: S1: fresh Chlamydomonas reinhardtii cells cultured to the rapid division phase are inoculated into a nutrient solution containing 0-2.5 g tris(hydroxymethyl)aminomethane, 4-18 g glucose monohydrate, 2-6 mg 6-benzyladenine, 2-6 mg 1-naphthaleneacetic acid, 0-10 g Porphyra extract and 1 portion of TAP medium without Tris per liter at a dry biomass of 0.15-0.25 g / L; S2: then adding the mixed solution into the reactor body (15) in the incubator body (10) for incubation; S3: The temperature and light intensity in the incubator body (10) and the stirring speed in the reactor body (15) are set to 9.5-10.5°C and 40-125 μE / m 2 / s and 400-2000rpm, continuous light culture for 120-170h, achieve a significant increase in the simultaneous production of α-linolenic acid and lutein in Chlamydomonas reinhardtii, and the yields of both are significantly higher than those under conventional culture conditions; S4: After the above-mentioned mixed liquid culture is completed, the algae liquid is discharged from the discharge pipe (16) below the reactor body (15). At this time, the cleaning component and the regulating component are used to quickly clean the inside of the reactor body (15).

8. The cultivation method of the microalgae enlargement cultivation device according to claim 7, characterized in that: The preparation process of the laver extract is as follows: chop fresh laver in the middle and late stages of maturity into pieces with a size of less than 0.5 mm, mix with an appropriate amount of pure water, and ultrasonically crush for 30 minutes, and sterilize to obtain the extract, wherein the mass volume ratio of the fresh laver to the pure water is 1:20-50.

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