Microalgae continuous culture method

Abstract

The invention relates to the technical field of microalgae continuous culture, and particularly discloses a microalgae continuous culture method, in the culture process, the optimal dilution rate Dopt can be determined through the following formula: the feasibility of the method is verified through experiments, and guidance is provided for large-scale continuous culture of microalgae.

Description

Technical Field

[0001] The present invention relates to the technical field of large-scale microalgae cultivation, and in particular to a high-efficiency continuous microalgae cultivation method based on optimized dilution rate. Background Art

[0002] Currently, microalgae cultivation primarily utilizes batch processes, requiring frequent reactor cleaning, sterilization, and inoculation, resulting in long production cycles and low efficiency. Batch cultures also experience significant diurnal fluctuations in environmental parameters, making it difficult to maintain optimal growth conditions and leading to significant quality variations between batches. In contrast, continuous culture significantly improves production efficiency and stability by maintaining a steady-state environment within the reactor through constant feeding and drainage.

[0003] However, existing continuous culture technologies mostly focus on single parameter regulation, failing to resolve the contradiction between dilution rate and average light intensity and nutrient limitation. There is still a lack of in-depth exploration of the theoretical modeling and parameter coordinated optimization mechanism of continuous culture processes, resulting in unsatisfactory practical application results and limiting the high-density and high-stability production of microalgae. Summary of the Invention

[0004] To address existing technical problems, the present invention establishes a dynamic mathematical model for continuous cultivation of microalgae, reveals the dynamic balance between biomass production and limiting factors during continuous cultivation, derives a dilution rate calculation formula through theoretical deduction, and combines it with experimental verification to confirm its feasibility. Furthermore, a more accurate method for calculating average light intensity is proposed, providing a theoretical basis and technical support for efficient, stable, and large-scale continuous production of microalgae.

[0005] In order to solve the technical problem of the present invention, the present invention provides a continuous culture method of microalgae, comprising the following steps: inoculating microalgae into a reactor, irradiating with a light source, and continuously replenishing culture fluid according to a set dilution rate to maintain a constant total liquid volume in the reactor;

[0006] Among them, the optimal dilution rate D opt It is determined by the following formula:

[0007]

[0008] Among them, I m The optimal average light intensity irradiating the algal cell surface is usually expressed in μmol / m 2 / s;

[0009] I k is the half-saturation coefficient of the effect of average light intensity on growth rate, usually expressed in μmol / m 2 / s;

[0010] C N,Ois the limiting nitrogen source concentration in the culture medium, and the unit can be mol / L, mmoL / L or g / L;

[0011] K N is the half-saturation coefficient of the effect of the limiting nitrogen source on the growth rate, and the unit can be mol / L, mmoL / L or g / L;

[0012] μ max The maximum specific growth rate of microalgae, usually expressed in days -1 .

[0013] In the scheme of the present invention, the optimal average light intensity I irradiated to the surface of algae cells is m The method is to cultivate microalgae under different average light intensities and select the average light intensity with the highest growth rate as the optimal average light intensity irradiated to the surface of algae cells.

[0014] In the scheme of the present invention, the half-saturation coefficient I of the average light intensity relative to the growth rate is k It is obtained by fitting the Monod equation curve of the specific growth rate of microalgae under different average light intensities. According to the fitted Monod equation, the average light intensity when half of the maximum specific growth rate is obtained is the half-saturation coefficient I of the limiting light source on the specific growth rate. k .

[0015] In the solution of the present invention, the limiting nitrogen source concentration C in the culture medium is N,O The method is to cultivate microalgae under different limiting nitrogen source concentration conditions and select the limiting nitrogen source concentration with the highest specific growth rate as the limiting nitrogen source concentration in the culture solution.

[0016] In the scheme of the present invention, the half-saturation coefficient K of the limiting nitrogen source relative growth rate is N,O It is obtained by fitting the Monod equation curve of the specific growth rate of microalgae under different limiting nitrogen source concentrations. According to the fitted Monod equation, the limiting nitrogen source concentration when half of the maximum specific growth rate is obtained is the half-saturation coefficient K of the limiting light source on the specific growth rate. N .

[0017] In the scheme of the present invention, the maximum specific growth rate μ of the microalgae is max That is, the maximum specific growth rate of microalgae monitored during the cultivation process.

[0018] In the solution of the present invention, when the reactor is a cylindrical reactor, the average light intensity I irradiated to the surface of the algae cells is av Satisfies the following formula:

[0019]

[0020] Wherein, I0 is the incident light intensity, usually in units of μmol / m 2 / s; C is the density of algal cells (also known as algal liquid concentration), usually in units of g / L; K a is the extinction coefficient of algal cells, m 2 / g; R is the radius of the reactor, which can be in units of mm, etc.; I k is the semi-saturation coefficient of the average light intensity on the growth rate, usually in units of μmol / m 2 / s; y is the height of any point on the circular arc segment of the reactor, which can be in units of mm, etc.

[0021] The average light intensity received by the microalgae is determined by the microalgae cell density, the microalgae extinction coefficient, the reactor radius and the incident light intensity. According to the real-time monitoring of each index, the formula can be brought into to accurately calculate the cell density at this time. According to the set average light intensity, the required incident light intensity can be inversely calculated, such as according to the optimal average light intensity I m irradiated to the surface of the algal cells, the formula can be used to inversely calculate the required incident light intensity, so as to accurately control the average light intensity.

[0022] In the present application, the extinction coefficient K a of the algal cells is determined by taking different concentrations of algal liquid, adjusting the incident light intensity, measuring the outgoing light intensity under different light paths, establishing a Lambert-Beer model, and then measuring a series of incident light intensities and outgoing light intensities of different algal liquid layer heights. The data obtained is obtained by non-linear curve fitting, and the Lambert-Beer model satisfies the following formula:

[0023]

[0024] Wherein, I AV is the average light intensity irradiated to the surface of the algal cells, I0 is the incident light intensity, usually in units of μmol / m 2 / s; K a is the extinction coefficient of the algal cells, m 2 / g; C x is the density of algal cells, g / L; L is the distance from the algal liquid to the light source, mm. The extinction coefficient calculation scheme is not only suitable for cylindrical reactors, but also suitable for flat plate reactors.

[0025] In the present application, in the culture process of the microalgae, the maximum yield P max under the optimal dilution rate is calculated by the following formula:

[0026]

[0027] Wherein, Y X / NIt is the nitrogen source conversion rate, that is, how many grams of algae cells are converted per mole of limiting nitrogen source, g / mol. During the cultivation of microalgae, the limiting nitrogen source is usually nitrate, etc.

[0028] As an improvement of the above technical solution, the microalgae is Chaetoceros algae.

[0029] As an improvement of the above technical solution, the microalgae is Chaetoceros muelleri.

[0030] As an improvement of the above technical solution, when the microalgae is Chaetoceros muelleri, the culture medium for continuous culture includes artificial seawater / seawater, F / 2 culture medium, bicarbonate, and metasilicate; the volume proportion of F / 2 culture medium in the culture medium is 1 to 5 ml / L; the concentration of bicarbonate in the culture medium is 0.01 to 0.08 mol / L, and the concentration of metasilicate is 0.1 to 0.4 mmol / L.

[0031] As an improvement of the above technical solution, when the microalgae is Chaetoceros muelleri, the pH of the culture solution is 8-9 and the temperature is 30-35°C.

[0032] The implementation of the present invention has the following beneficial effects:

[0033] 1. The present invention systematically integrates the light attenuation effect, the dynamic equilibrium mechanism of limiting nutrient concentration and dilution rate to construct a kinetic model for continuous microalgae culture. Based on the mathematical expression of cell density and yield, the mathematical expression of cell density and yield is further derived. The optimal dilution rate for maximum yield is theoretically calculated using the second-order derivative method, providing a quantifiable theoretical framework for large-scale continuous culture.

[0034] 2. After calculating the optimal dilution rate, a small-scale experiment was conducted to verify that the dilution rate was 0.8 days. -1 The theoretical optimal yield at 0.4 days effectively balances cell growth and washout risk. In contrast, previous studies have mostly used low dilution rates (such as 0.4 days -1 ), failed to fully utilize the metabolic potential of Chaetoceros muelleri, while the cell density maintained by too high a dilution rate was too low, and the volume required to be processed every day was too large, resulting in low economic benefits.

[0035] 3. The present invention uses the double integral method to propose a method for calculating the average light intensity of a cylindrical reactor (including tubular and bubble reactors) under external parallel light illumination conditions. The average light intensity received by the microalgae is determined by the microalgae cell density, microalgae extinction coefficient, reactor radius, and incident light intensity. The cell density at this time can be accurately calculated by substituting various indicators monitored in real time into the formula. The required incident light intensity can also be inversely calculated based on the set average light intensity, thereby achieving precise control of the average light intensity.

[0036] 4、Traditional batch culture fluctuates greatly in various culture conditions between batches, and it is difficult to maintain the optimal growth conditions, resulting in long production cycle and poor stability. The continuous culture mode proposed in this scheme can realize accurate regulation of pH, temperature and light intensity, and optimize the proportion of limiting nutrients in the culture medium. Culturing under optimal conditions of various culture conditions can maintain a high cell density. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The average light intensity plane calculation model for the cylindrical reactor;

[0038] Figure 2 The growth curve of M. muelleri under different concentrations of sodium nitrate;

[0039] Figure 3 The Monod equation curve of M. muelleri under different concentrations of sodium nitrate;

[0040] Figure 4 The fitting curve of the extinction coefficient of M. muelleri;

[0041] Figure 5 The growth curve of M. muelleri under different average light intensities;

[0042] Figure 6 The Monod equation curve of M. muelleri under different average light intensities;

[0043] Figure 7 The relationship between dilution rate and dry weight and cell number of M. muelleri after reaching stability in continuous culture;

[0044] Figure 8 The relationship between dilution rate and growth rate of M. muelleri after reaching stability. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application will be further described in detail below with specific embodiments.

[0046] The present application provides a continuous culture method of microalgae. In the culture method, microalgae are inoculated into a reactor, irradiated with a light source, and continuously supplemented with culture medium at a set dilution rate to maintain a constant total liquid volume in the reactor. By modeling, the optimal concentration of limiting nutrients is optimized, the optimal average light intensity is optimized, and a more accurate average light intensity calculation method is proposed. The optimal dilution rate is determined and the theoretical maximum yield is calculated, and the dynamic balance law of biomass production and limiting factors in continuous culture is revealed. The present application provides a theoretical basis and technical support for efficient, stable and large-scale production of microalgae. The optimal dilution rate of continuous culture is determined, and the feasibility of the method of the present application is verified by experiments. Specifically as follows:

[0047] (1) Modeling of microalgae continuous culture process

[0048] In a continuous bioreactor, algal liquid is continuously removed from the culture vessel, and fresh culture liquid enters the culture reactor at a constant flow rate, which is equal to the volume flow rate of the algal liquid flowing out of the reactor, so that the total liquid volume in the reactor is constant. In steady-state operation, the algal cell density in the reactor is closely related to the average light intensity and the limiting nutrient concentration C. N,O The relationship satisfies formula (1).

[0049]

[0050] Where C x is the algal cell density, g / L; Y X / N is the conversion rate, that is, how many grams of algae cells are converted per mole of nitrate ions, C N,O is the limiting nitrogen source concentration in the culture medium, usually the nitrate ion concentration; D is the dilution rate, K N I is the half-saturation coefficient of the effect of limiting nitrogen source on growth rate; m is the optimal average light intensity irradiated to the surface of microalgae cells, I k is the half-saturation coefficient of the effect of average light intensity on growth rate, μ max is the maximum specific growth rate of microalgae. This formula describes the rate and amount of biomass production in a continuous bioreactor through the material balance of cell biomass and dissolved limiting nutrients.

[0051] For the convenience of calculation, the coefficient of light intensity is set to m. Through experimental calculation, m is a constant, and its expression is as shown in formula (2).

[0052]

[0053] The microalgae cell yield P is equal to the cell density multiplied by the dilution rate. Substituting it into formula (1), we can obtain the calculation formula (3) for the yield P.

[0054]

[0055] The dilution rate D must be less than the maximum specific growth rate μ max Otherwise, washing out will occur. Set this relationship coefficient to a, and let the dilution rate D = a × μ max , where 0<a<m<1, and then the calculation expression (3) of the yield P is simplified to obtain the formula (4).

[0056]

[0057] (2) Derivative process of maximum yield of Chaetoceros

[0058] According to the above-derived functional relationship expression (4) between yield and a, the derivative of both sides of the equation with respect to a is obtained, and the derivative result is shown in formula (5).

[0059]

[0060] Let the first-order derivative be zero and further simplify formula (5) to obtain formula (6).

[0061]

[0062] When 1>m>a>0, Discard results greater than 1.

[0063] Calculate the second-order derivative of P with respect to a. The calculation result of the second-order derivative is as shown in formula (7).

[0064]

[0065] Since 1>m>a>0, the second-order derivative is less than zero and the function reaches a maximum value.

[0066] Then when When P reaches its extreme value, theoretically the maximum yield P can be obtained. max , specifically as formula (8).

[0067]

[0068] D=a×μ max , similarly, when When the dilution rate D reaches its maximum value D opt , as shown in formula (9).

[0069]

[0070] (3) Average light intensity integration method

[0071] Currently, most research on light intensity focuses on the effect of incident light intensity on microalgae growth. Appropriate light intensity can promote biomass growth, while both high and low light intensities can inhibit it. Since light in the reactor continuously attenuates, the actual photon utilization rate of the microalgae cells decreases. Therefore, the light intensity that directly affects microalgae growth is not the incident light intensity, but the average light intensity. Currently, most methods for calculating average light intensity use flat-plate reactors. Using the same method for cylindrical reactors such as bubble reactors and tubular reactors results in errors. Therefore, this study uses double integrals to provide a new method for calculating average light intensity in cylindrical reactors.

[0072] The calculation principle is based on the Lambert-Beer law, which can be described as follows: when a beam of parallel monochromatic light passes vertically through a uniform non-scattering absorbing material, the relationship between the intensity of the material's light absorption and the concentration of the absorbing material and the thickness of its liquid layer. The specific mathematical expression is shown in formula (10):

[0073] I Z =I0e -KcCxL (10)

[0074] Where, I z is the intensity of the emitted light (μmol / m 2 / s); I0 is the incident light intensity (μmol / m 2 / s); L is the distance light travels in the algae solution (mm); K c is the extinction coefficient of microalgae (m 2 / g), C x is the cell density of microalgae (g / L).

[0075] The top cross-sectional view of the reactor is used to establish Figure 1 According to the mathematical model, the average light intensity of the upper and lower semicircles of the reactor is equal according to the principle of symmetry. For the convenience of calculation, this method only calculates the total light intensity received by the upper semicircle. In the final calculation, the calculated total light intensity is divided by the illuminated area of ​​the upper semicircle. The obtained average light intensity can represent the average light intensity received by the entire reactor.

[0076] Establish a plane rectangular coordinate system with the left vertex of the reactor as the coordinate origin O, then the semicircle equation expression is (xR) 2 +y 2 =R 2 (y>0), let two points A(x0, y0) and B(2R-x0, y0) on the circle be parallel to the x-axis. According to the Lambert-Beer law, light gradually attenuates during the propagation of algae liquid, but no attenuation occurs in the air medium. Therefore, the initial incident light intensity of the left arc segment is I0. Then the light intensity expression of any point C on line segment AB is as shown in formula (11), where L is the horizontal coordinate of point C on AB.

[0077]

[0078] The total light intensity I of line segment AB can be obtained by integrating from A to B. AB As shown in formula (12), where 0<x0<2R.

[0079]

[0080] Take the length of the small integral segment as dy, and integrate the function y from 0 to R to calculate the total light intensity of the semicircle as shown in formula (13), where

[0081]

[0082] The average light intensity is the total light intensity of the semicircle divided by the area of ​​the semicircle. av The calculation results are shown in formula (14).

[0083]

[0084] When AB moves to the x-axis, x0=0, incident light I0=incident light from the light source, outgoing light I=outgoing light after attenuation from the light source, and by substituting the Lambert-Beer light attenuation equation into it, the cell density C can be obtained. x , back to the average light intensity calculation formula (14) to get the average light intensity I av The relationship between the incident light intensity I0 and the illumination intensity during the microalgae cultivation process is thus maintained at the optimal condition.

[0085] (4) Process of determining various parameters of the continuous culture model

[0086] If we want to find the maximum yield P achieved by formula (8) max A series of experiments are needed to determine the cell density C during continuous culture. X and the optimum average light intensity I m and limiting nutrient concentration C N,O The various parameters in the relational expression (1) are summarized as follows:

[0087] 1)I m is the optimal average light intensity irradiated to the surface of algae cells. av When culturing microalgae under certain conditions, it can be concluded that the specific growth rate of algae cells is the highest when the average light intensity is specific, and the average light intensity value is I m ;

[0088] 2)I k The half-saturation coefficient of the effect of average light intensity on the growth rate is obtained by fitting the specific growth rate of microalgae under different average light intensities to obtain the Monod equation curve of microalgae under different average light intensities. The half-saturation coefficient of the effect of average light intensity on the growth rate is obtained by taking the average light intensity value at half the maximum specific growth rate. k ;

[0089] 3) C N,OC is the limiting nitrogen source concentration in the culture medium. Nitrate is usually used as the limiting nitrogen source. Specifically, microalgae can be cultured under different sodium nitrate concentrations. It is found that the specific growth rate of algae cells is the highest when the sodium nitrate concentration is a certain concentration. This concentration is C N,O ;

[0090] 4)K N is the half-saturation coefficient of the effect of the limiting nitrogen source on the specific growth rate. Taking sodium nitrate as the limiting nitrogen source as an example, the Monod equation curve of microalgae under different sodium nitrate concentrations can be obtained after fitting based on the specific growth rate of microalgae under different sodium nitrate concentrations. The sodium nitrate concentration at which half of the maximum specific growth rate is obtained is the half-saturation coefficient K of the effect of the limiting light source on the specific growth rate. N ;

[0091] 5)Y X / N is the nitrogen source conversion rate, that is, how many grams of algae cells are converted per mole of limiting nitrogen source. The nitrogen source is usually nitrate ions. For example, taking Chaetoceros muelleri as an example, the nitrogen source conversion rate Y can be obtained according to the following formula (15): X / N =221g cells / molNO3 - ;

[0092]

[0093] 6)μ max The maximum specific growth rate of microalgae monitored during cultivation.

[0094] (5) Calculation of the theoretically optimal dilution rate and maximum yield

[0095] According to the derivation results of the above formula and the determination results of the various parameters in (4), the optimal dilution rate D can be calculated by substituting it into the formula opt , and the maximum algal cell density Cx and the maximum yield P under the optimal dilution rate conditions max .

[0096] The continuous culture of Chaetoceros muelleri is taken as an example to illustrate and verify the culture method of the present invention.

[0097] It should be noted that:

[0098] 1) The culture medium for continuous culture of Chaetoceros muelleri can be prepared using seawater or artificial seawater, wherein F / 2 culture medium, bicarbonate, metasilicate, etc. are added. The carbonate salt can be sodium bicarbonate, etc.; the metasilicate can be sodium metasilicate or its hydrate. The composition of artificial seawater is shown in Table 1, and the composition of F / 2 culture medium concentrate is shown in Table 2. The limiting nitrogen source is mainly introduced by sodium nitrate in F / 2 culture medium. The volume proportion of F / 2 culture medium in the culture medium can be 1-5 ml / L, and can be 1 ml / L, 2 ml / L, 3 ml / L, 4 ml / L, 5 ml / L, etc., but is not limited to this. It is set based on the optimal limiting nitrate concentration. Preferably, the volume proportion of F / 2 culture medium is 2 ml / L. The concentration of bicarbonate in the culture medium is 0.01 to 0.08 mol / L, and illustratively can be 0.01 mol / L, 0.02 mol / L, 0.04 mol / L, 0.06 mol / L, 0.08 mol / L, etc., but not limited thereto. Preferably, the concentration of bicarbonate in the culture medium is 0.04 mol / L; the concentration of metasilicate in the culture medium is 0.1 to 0.4 mmol / L, and illustratively can be 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, etc., but not limited thereto. Preferably, the concentration of metasilicate in the culture medium is 0.2 mol / L.

[0099] Table 1 Composition of artificial seawater

[0100]

[0101] Table 2 Composition of F / 2 culture medium concentrate

[0102]

[0103]

[0104] 2) During the culture process, the pH of the culture medium is controlled between 8.0 and 9.0, illustratively, 8, 8.5, 9, etc., but not limited thereto. Preferably, the pH of the culture medium is 8.5. The culture temperature is controlled between 30 and 35°C, illustratively, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, etc., but not limited thereto. Preferably, the temperature of the culture medium is 34°C.

[0105] 3) During the culture process, the incident light intensity was adjusted according to the cell density of Chaetoceros hornii, and different average incident light intensities (such as 60, 90, 120, 150 and 180 μmol / m 2 / s, etc.), culture was carried out, and the optimal average incident light intensity was selected according to the maximum specific growth rate.

[0106] 4) Continuous culture dilution rate is 0.2-1.0 day-1 , set different dilution rate, get the best dilution rate, through and the best dilution rate derived by formula comparison, verify the accuracy and feasibility of the application scheme.

[0107] Example 1 NaNO3 concentration on the influence of Mou's Chaetoceros and parameter determination

[0108] In order to optimize the optimal concentration of Mou's Chaetoceros, improve the growth rate of Mou's Chaetoceros and cell density and biomass components, the specific growth rate under different sodium nitrate concentration was determined by experiment, and the Monod equation was fitted according to the specific growth rate, and the half saturation coefficient K of the influence of limiting nitrogen source concentration on specific growth rate was calculated N .

[0109] In this experiment, single factor test method was used to screen the concentration of sodium nitrate in the culture solution. Based on the artificial seawater medium without nitrogen nutrient salt, 10 groups of nitrogen-free medium were added with different nitrogen source (sodium nitrate) concentrations (0, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 6 and 8 mmol / L) F / 2 medium to culture Chaetoceros, and 0.04 g / L of algal species was inoculated for culture in 100 mL conical flask (working volume 50 mL), the light mode was continuous light with an average light intensity of 120 μmol / m 2 / s, the pH was stabilized at about 8.5, the temperature was controlled at 34℃, the culture period was 72h, the microalgae biomass from 0h to 72h was determined, 3 groups of parallel experiments were set for each condition, and the data was recorded every 12 hours. The cell growth condition was measured every 12h in the first two days, and every 24h in the third day.

[0110] The growth of Chaetoceros cultured under different sodium nitrate concentrations is shown in Figure 2 From the figure, it can be seen that when the sodium nitrate concentration is ≥2 mmol / L, the algal cells can achieve the maximum specific growth rate, and higher gradient of sodium nitrate concentration does not have obvious improvement on the growth rate of algal cells, therefore, the optimal limiting nitrogen source concentration C N，0 =2 mmol / L=0.028 g / L. At this time, the addition amount of F / 2 medium in the culture solution is 2 mL / L.

[0111] According to the maximum specific growth rate of 36h, the Monod equation curve of Chaetoceros under different sodium nitrate concentrations can be obtained by plotting and fitting, as shown in Figure 3 From the fitted Monod equation expression, it can be seen that the half saturation coefficient K N of the limiting sodium nitrate concentration when the maximum specific growth rate is half is 0.096 mmol / L=0.001344 g / L.

[0112] Example 2 Extinction coefficient K of Chaetoceros muelleri a Determination

[0113] Light intensity has different penetration capabilities in culture solutions with varying microalgae concentrations and culture depths. Therefore, calculating the average light intensity received by the microalgae requires measuring the degree of light attenuation under different conditions. This experiment employed the Lambert-Beer model to measure light attenuation. The specific formula for average light intensity is shown in Equation 16:

[0114]

[0115] Among them I AV is the average light intensity irradiated to the algal cell surface, I0 is the incident light intensity, μmol / m 2 / s;K a is the extinction coefficient of algae cells, m 2 / g; C is the algae cell density, g / L; L is the distance from the algae liquid to the light source, mm.

[0116] Algal suspensions of varying cell densities were placed in 2000 mL beakers. An LED light source was placed at the bottom of the beaker to adjust the light intensity. Before changing the light intensity, the intensity of light transmitted without the addition of algal solution (defined as the initial light intensity) was measured, and the outgoing light intensity was measured at different light paths. The Lambert-Beer model was used to measure the incident and outgoing light intensities at various liquid layer heights (2.5, 5, and 10 cm). The measured data were fitted with a nonlinear curve using Origin software, and the fitting results are shown in Figure 2. Figure 4 As shown, the extinction coefficient of Chaetoceros muelleri is obtained as K a 0.115m 2 / g.

[0117] Example 3 Effect of average light intensity on Chaetoceros muelleri and parameter determination

[0118] After measuring the light attenuation, the growth state of Chaetoceros was monitored with different average light intensities as a single variable. In this experiment, five groups of different average light intensities (60, 90, 120, 150 and 180 μmol / m 2Chaetoceros muelleri was cultured at 4000 rpm for 3 minutes in the logarithmic growth phase. The supernatant was removed and the algal slurry was inoculated into 200 mL of culture medium at a cell density of 0.04 g / L. Per liter of culture medium, 3.36 g of bicarbonate, 0.03 g of sodium metasilicate nonahydrate (Na2SiO3·9H2O), and 2 mL of F / 2 culture medium concentrate were added. The remainder was supplemented with artificial seawater. Cultures were performed for 3 days, maintaining a stable pH around 8.5. The temperature was controlled at 34°C. Three parallel experiments were performed for each condition, and data were recorded every 12 hours. Cell growth was measured every 12 hours for the first two days and every 24 hours on the third day.

[0119] The growth of Chaetoceros spp. under different light intensities Figure 5 As shown, the Monod equation curve of Chaetoceros under average light intensity can be obtained by plotting and fitting the maximum specific growth rate of Chaetoceros under 36 hours of growth. Figure 6 As shown, the half-saturation coefficient I of the average light intensity of Chaetoceros can be obtained based on the Monod equation curve. k =35.51 μmol / m 2 / s.

[0120] In addition, from Figure 5 The average light intensity is 120 μmol / m 2 / s, the specific growth rate of algal cells is the highest, and when the average light intensity is less than 120 μmol / m 2 / s, the light level is too low, the growth and accumulation of microalgae will be significantly restricted. Due to insufficient light intensity, algae cells cannot obtain enough light sources, resulting in the algae cell specific growth rate gradually increasing with the increase of average light intensity. When the average light intensity is greater than 120μmol / m 2 / s, the growth of algae cells is inhibited due to the high average light intensity. When the light intensity exceeds the tolerance range of algae cells, the PSII complex will be damaged, which will lead to photoinhibition and a significant decrease in biological productivity. Therefore, the average light intensity is 120μmol / m 2 / s is most conducive to the growth of Chaetoceros muelleri, that is, I m =120 μmol / m 2 / s.

[0121] According to the above examples 1-3, the following Chaetoceros algae were obtained:

[0122] I m =120 μmol / m 2 / s,I k =35.51 μmol / m 2 / s,C N,O=2mmol / L,K N =0.096mmol / L,K a

[0123] =0.115m 2 / g,Y X / N =221g cells / mol NO3 - ;

[0124] In addition, the maximum specific growth rate μ of Chaetoceros monitored during the actual cultivation process was max =1.4day -1 .

[0125] Then, the optimal dilution rate D can be calculated based on the derivation results of the above formula (1-14) and the determination results of various parameters. opt The maximum algal cell density Cx and the maximum yield P under the conditions max , the calculation results are as follows

[0126] As shown in Formula 17, Formula 18, Formula 19, and Formula 20.

[0127]

[0128] According to the calculation results, the dilution rate is maintained at 0.8492day during the continuous culture process. -1 The theoretical maximum yield can be achieved when the system can maintain a dynamic cell density of 0.3641 g / L.

[0129] Based on the calculation results, an example is set for verification, as shown in Example 4.

[0130] Example 4 Experimental study on the effect of dilution rate of continuous culture on the growth of Chaetoceros muelleri

[0131] In the indoor continuous culture experiment, the single-factor test method was used to test different dilution rates. After 3-5 culture volumes were cultured at each dilution rate to reach stability, the final stable cell density and dry weight were measured.

[0132] The indoor reactor used a small-scale fermentation tank for the experiment, with a culture scale of 5 L. The culture medium conditions were as follows: 3.36 g bicarbonate, 0.03 g sodium metasilicate (Na2SiO3·9H2O), and 2 ml F / 2 culture medium concentrate were added to each liter of culture medium, and the balance was supplemented with artificial seawater. LED light source was used for supplementary lighting. According to the optical path of the reactor, the extinction coefficient of Chaetoceros, and the cell density during continuous culture, the average light intensity I was introduced. av Formula (14) was used to calculate and adjust the incident light intensity in real time so that the average light intensity irradiated on the algae cell surface reached 120 μmol / m 2s, ensure pH stable in the optimum range 8.5, temperature control at 34℃, dilution rate gradient set to 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 day -1 , the absorbance and cell density were measured every day during the culture to monitor the growth state of the Chaetoceros, and the specific experimental results are shown in Figure 7 . It should be noted that: because the growth state after reaching the stable state of continuous culture is not related to the initial inoculation density, therefore the initial inoculation density in this experiment is not required to be explicitly unchanged.

[0133] According to the experimental results, the growth rate under each dilution rate condition was calculated after reaching the stable state, and the summary is shown in Figure 8 . According to the summary results, the culture efficiency of 0.8 day -1 (4L of liquid change per day) can reach 0.216g / L / day, which is significantly higher than the culture method of other dilution rates in the same batch, and the dry weight of 0.27g / L and the cell number of 580×10 4 / ml were monitored after reaching the stable state, compared with 0.134g / L / day of batch culture under the same conditions, the growth efficiency of continuous culture can be improved by 61%. In this experiment, according to the lighting lamp of 6.7W, the reactor volume of 5.0L, the dilution rate of 0.8 day -1 , the culture efficiency of 0.216g / L / day is calculated, and the conversion rate of microalgae is 148.9kWh / kg, which is significantly lower than the current industrial 180kWh / kg.

[0134] According to the experimental results, the maximum yield can be obtained when the dilution rate is 0.8 day -1 , which is close to the optimum dilution rate of 0.8492 day -1 calculated, which shows that the above derived formula has guiding significance for actual production.

[0135] The above takes Chaetoceros as an example to illustrate the feasibility of the dilution rate calculation method of the present application, but is not limited thereto. The culture method of the present application is also applicable to the continuous culture of other microalgae.

[0136] The above is the preferred embodiment of the application, and it should be pointed out that for ordinary skilled persons in the technical field, some improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements are also considered to be within the protection scope of the present application.

Claims

1. A method for continuous cultivation of microalgae, characterized in that: The following steps are involved: The microalgae are inoculated into the reactor, irradiated with a light source, and the culture solution is continuously replenished according to the set dilution rate to maintain a constant total liquid volume in the reactor; Optimal dilution rate D opt It is determined by the following formula: Among them, I m is the optimal average light intensity irradiating the algal cell surface; I k is the half-saturation coefficient of the effect of average light intensity on growth rate; C N,O is the limiting nitrogen source concentration in the culture medium; K N is the half-saturation coefficient of the effect of limiting nitrogen source on growth rate; μ max is the maximum specific growth rate of microalgae.

2. The method for continuous cultivation of microalgae according to claim 1, wherein: The optimum average light intensity I m The method is to cultivate microalgae under different average light intensities and select the average light intensity with the highest growth rate as the optimal average light intensity irradiated to the surface of algae cells. The half-saturation coefficient I of the effect of the average light intensity on the growth rate k It is obtained by fitting the Monod equation curve of the specific growth rate of microalgae under different average light intensities. According to the fitted Monod equation, the average light intensity when half of the maximum specific growth rate is obtained is the half-saturation coefficient I of the limiting light source on the specific growth rate. k .

3. The method for continuous cultivation of microalgae according to claim 1, wherein: The limiting nitrogen source concentration C in the culture medium N,O The method is to cultivate microalgae under different limiting nitrogen source concentrations and select the limiting nitrogen source concentration with the highest specific growth rate as the limiting nitrogen source concentration in the culture solution; The half-saturation coefficient K of the limiting nitrogen source on the growth rate N,O It is obtained by fitting the Monod equation curve of the specific growth rate of microalgae under different limiting nitrogen source concentrations. According to the fitted Monod equation, the limiting nitrogen source concentration when half of the maximum specific growth rate is obtained is the half-saturation coefficient K of the limiting light source on the specific growth rate. N .

4. The method for continuous cultivation of microalgae according to claim 1, wherein: When the reactor is a cylindrical reactor, the average light intensity I av Satisfies the following formula: Where I0 is the incident light intensity; C x is the algal cell density; K a is the extinction coefficient of algae cells; R is the reactor radius; I k is the half-saturation coefficient of the effect of average light intensity on growth rate; y is the height of any point in the reactor arc segment.

5. The method for continuous cultivation of microalgae according to claim 4, wherein: The algae cell extinction coefficient K a The Lambert-Beer model is established by taking algae liquid of different concentrations, adjusting the incident light intensity, and measuring the outgoing light intensity at different light paths. The incident and outgoing light intensities at a series of different algae liquid layer heights are then measured and the obtained data are fitted with a nonlinear curve. The Lambert-Beer model satisfies the following formula: Among them I AV is the average light intensity irradiated to the surface of algae cells, I0 is the incident light intensity; K a is the extinction coefficient of algae cells; C x is the algae cell density; L is the distance from the algae liquid to the light source.

6. The method for continuous cultivation of microalgae according to any one of claims 1 to 5, wherein: During the cultivation of the microalgae, the maximum yield P under the optimal dilution rate condition is max The calculation is done using the following formula: Among them, Y X / N is the nitrogen source conversion rate, that is, how many grams of algal cells are converted per mole of limiting nitrogen source.

7. The method for continuous cultivation of microalgae according to any one of claims 1 to 6, wherein: The microalgae is Chaetoceros.

8. The method for continuous cultivation of microalgae according to claim 7, wherein: The microalgae is Chaetoceros muelleri.

9. The method for continuous cultivation of microalgae according to claim 8, wherein: The culture solution includes artificial seawater / seawater, F / 2 culture medium, bicarbonate, and metasilicate; the volume proportion of the F / 2 culture medium in the culture solution is 1 to 5 ml / L; the concentration of bicarbonate in the culture solution is 0.01 to 0.08 mol / L, and the concentration of metasilicate is 0.1 to 0.4 mmol / L.

10. The method for continuous cultivation of microalgae according to claim 9, wherein: The pH of the culture solution is 8-9, and the temperature is 30-35°C.

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