Method for synergistically regulating and controlling microbial growth in stages based on light / carbon / nutrient substances

Abstract

The invention discloses a method for synergistically regulating and controlling microbial growth in stages based on light / carbon / nutrient substances, which comprises the following steps: according to the growth cycle of photosynthetic microorganisms, the photosynthetic microorganisms are cultured by changing control parameters, and the control parameters are one or more of dynamic regulation illumination intensity, nutrient element or carbon source supply. According to the method disclosed by the invention, through dynamic illumination and staged nutrient element and carbon source supply, the light energy and nutrition utilization efficiency in the photosynthetic microorganism culture process is remarkably improved. Compared with an existing constant illumination and traditional nutrition supply mode, the light intensity, the light period and the nutrition supply strategy can be dynamically adjusted according to the actual requirements of microorganisms in different growth stages, light inhibition and nutrient waste are avoided, rapid growth of the microorganisms and accumulation of target products are effectively promoted, and therefore the culture yield and efficiency are remarkably improved. The method provided by the invention can be widely applied to the fields of environmental governance, biofuel, medicine, food and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of photosynthetic microorganism cultivation, and in particular relates to a method for coordinating microbial growth in stages based on light / carbon / nutrients. Background Art

[0002] Photosynthetic microorganisms absorb light energy through photosynthesis and convert inorganic substances such as carbon dioxide into bioproducts such as carbohydrates, lipids, and proteins, achieving biomass accumulation and energy conversion. In the process of achieving photosynthetic autotrophic growth, the basic substrates required by microorganisms include gaseous carbon dioxide (CO2), essential nutrients such as nitrogen and phosphorus in the liquid phase, and light to provide energy for metabolic reactions.

[0003] Among them, carbon is one of the most important elements in microalgae cells, accounting for about 50% of the cell dry weight, so efficient carbon supply is particularly critical. Under industrial conditions, CO2 often comes from flue gases emitted by coal-fired power plants, cement plants, chemical plants, etc. The CO2 concentration of this type of flue gas is as high as 10-20%, which has certain application potential, but the traditional continuous gas supply method is prone to excessive CO2 escape and resource waste. At the same time, liquid nutrients such as nitrogen, phosphorus, sulfur, magnesium and trace elements are necessary components for forming microbial cell structure and maintaining their metabolic activities. In order to promote the rapid proliferation of microalgae, existing technologies also widely use means such as adjusting nutrient ratios, supplementing nutrient solutions, and controlling the pH and temperature of the culture solution to optimize the culture environment. However, due to the significant differences in the types and amounts of nutrients required by microorganisms during the adaptation period, logarithmic growth period and stable period, a single or constant mode of addition is difficult to achieve efficient regulation.

[0004] During the acclimatization phase, microalgae cells primarily adapt to their external environment, with relatively low nutritional requirements. Supplying high nitrogen concentrations prematurely during this phase can lead to overproduction of pigments (such as chlorophyll), resulting in a light-shading effect, reduced light penetration, and compromised photosynthetic efficiency in subsequent stages. After entering the logarithmic growth phase, cells actively proliferate, significantly increasing their demand for elements like nitrogen and phosphorus. Appropriate nutritional supplementation can significantly promote biomass accumulation. During the stationary or induction phase, reducing nitrogen supply can stimulate a shift in cellular metabolism toward lipid synthesis, increasing the accumulation rate of lipid products.

[0005] On the other hand, the light as the energy source of photosynthesis has a direct impact on the growth of microorganisms and product synthesis in the way of supply. Long-term use of constant light is easy to cause light intensity to be too high in some growth stages, resulting in photoinhibition; or the light supply is insufficient in the rapid cell division period, limiting the photosynthetic rate, and ultimately inhibiting growth and product formation. When the nutrients such as nitrogen and phosphorus are sufficient, microalgae will synthesize more photosynthetic pigments, thereby enhancing the light absorption capacity; if the light intensity is insufficient, the absorbed nutrients cannot be fully utilized, which shows that the nutrient utilization rate is reduced and the growth rate is slowed down; on the contrary, if the light intensity is too high, when the nutrient supply is insufficient, the excess energy cannot be effectively utilized, resulting in photooxidative stress. Therefore, it is urgent and meaningful to explore the dynamic coupling and matching between light intensity, carbon, nitrogen, phosphorus and other nutrients to maintain efficient photosynthetic efficiency and metabolic level in each growth stage. SUMMARY

[0006] The present application aims to at least solve one of the technical problems in the related art. To this end, the main purpose of the present application is to provide a method for phase-based synergistic regulation of microorganism growth based on light / carbon / nutrients, which can improve the growth rate and target product accumulation efficiency of photosynthetic microorganisms, and aims to solve the problems of low gas-liquid substrate utilization efficiency, low photosynthetic growth rate, low light energy utilization rate and low nutrient supply efficiency in the prior art.

[0007] The purpose of the present application is achieved by the following technical solutions: A method for phase-based synergistic regulation of microorganism growth based on light / carbon / nutrients, comprising the following steps: according to the growth cycle of photosynthetic microorganisms, cultivating photosynthetic microorganisms by changing control parameters, the control parameters being dynamic adjustment of the concentration of nutrients.

[0008] The nutrients are one or more of nitrogen, phosphorus, sulfur, magnesium and trace elements.

[0009] In the method of the present application, different growth cycles of different photosynthetic microorganisms are used to determine the optimal control parameters and the corresponding best parameter conditions, so as to improve the utilization efficiency of light energy and nutrients in the cultivation process of photosynthetic microorganisms.

[0010] The growth cycle of the photosynthetic microorganism includes lag phase (0-2 days), exponential phase (2-7 days) and stationary phase (5-15 days), such as Figure 7As shown, photosynthetic microorganisms have significantly different requirements for energy factors such as light, carbon sources, and nutrients (such as nitrogen and phosphorus) at different growth stages. During the adaptation phase (the hysteresis phase in the figure), cells are in a stage of environmental response and metabolic activation, with low tolerance to light intensity and nutrient concentration. A low-input strategy is recommended to avoid photoinhibition and nutrient stress. The exponential growth phase is a critical stage for rapid microbial proliferation. During this period, the demand for substrates such as light, dissolved CO2, and nitrogen and phosphorus reaches its peak. Enhanced light supply and precise nutrient and carbon source replenishment are necessary to support rapid biomass growth. During the stationary phase or product accumulation phase, cellular metabolism shifts to the synthesis of energy storage products (such as lipids), and the demand for nutrients and light decreases. Appropriate induction strategies such as nitrogen limitation and low light levels can help promote the accumulation of target products.

[0011] In some embodiments, the control parameters further include dynamically adjusting light intensity.

[0012] In some embodiments, the control parameters further include dynamically adjusting the concentration of the carbon source.

[0013] In certain embodiments, the concentration of the nutrient element is 0.01-0.5 g / L in the hysteresis phase of the microalgae biomass, 0.5-1.5 g / L in the exponential phase of the biomass, and 0.25-2.0 g / L in the stationary phase of the biomass.

[0014] In some embodiments, the light intensity concentration during the hysteresis period is 30-150 μmol / m 2 ·s, and the concentration in the exponential phase is 100-1000 μmol / m 2 ·s, and the concentration in the stable period is 50-500 μmol / m 2 · s.

[0015] In certain embodiments, the carbon source concentration (gas phase) is 1-5% in the hysteresis phase, 5-20% in the exponential phase, and 2-15% in the stationary phase.

[0016] In certain embodiments, the carbon source includes but is not limited to gaseous CO2, and the photosynthetic microorganism includes but is not limited to microalgae.

[0017] As the same inventive concept, the present application also provides the application of the method of phased coordinated regulation of microbial growth based on light / carbon / nutrients in the fields of environmental governance, biofuels, medicine and food.

[0018] Compared with the prior art, the present invention has at least the following advantages: The method of the present invention significantly improves the efficiency of light energy and nutrient utilization during the cultivation of photosynthetic microorganisms through dynamic illumination and phased nutrient and carbon source supply. Compared to existing constant illumination and traditional nutrient supply models, this method dynamically adjusts light intensity, nutrient supply, and carbon source supply strategies based on the actual needs of microorganisms at different growth stages, avoiding photoinhibition and nutrient waste. It effectively promotes rapid microbial growth and accumulation of target products, thereby significantly improving culture yield and efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the specific embodiments of the present invention, the following briefly introduces the drawings required for the specific embodiments or the description of the prior art.

[0020] Figure 1 This is a diagram showing the stage-by-stage regulation of sodium nitrate during the cultivation process in the method provided in Example 1 of the present invention; Figure 2 This is a trend graph of the biomass concentration of microalgae cultured by the method in Example 1 and Example 1 of the present invention; Figure 3 This is a diagram showing the nitrogen source concentration and light intensity control strategy in different stages during the cultivation process in the method provided in Example 2 of the present invention; Figure 4 This is a trend diagram of the biomass concentration of microalgae cultured by the method in Example 2 and Example 2 of the present invention; Figure 5 This is a diagram showing the control strategy for nitrogen source concentration, light intensity, and carbon source concentration in different stages during the cultivation process in the method provided in Example 3 of the present invention; Figure 6 This is a trend graph of the biomass concentration of microalgae cultured by the methods in Example 3 and Example 2 of the present invention; Figure 7 This is the energy synergy demand and matching relationship of the microbial growth cycle in the method provided by the present invention. DETAILED DESCRIPTION

[0021] The present invention will be further described in detail below with reference to the accompanying drawings and examples. The following examples are merely illustrative and non-restrictive, and should not be used to limit the scope of protection of the present invention.

[0022] When expressing a certain amount, concentration or other value or parameter in the form of a range, preferred range, or preferred upper and lower numerical limits, it should be understood that it is equivalent to specifically disclosing any range by combining any pair of upper range limits or preferred numerical values ​​with any lower range limit or preferred numerical value, without considering whether the range is specifically disclosed. Unless otherwise indicated, the numerical range values ​​listed herein include the endpoints of the range and all integers and fractions within the range.

[0023] Unless otherwise indicated, all percentages, parts, ratios, etc. herein are by weight.

[0024] The materials, methods, and examples herein are illustrative and, unless otherwise indicated, are not to be construed as limiting.

[0025] Example 1 This embodiment provides a method for coordinating the growth of microorganisms in stages based on light / carbon / nutrients, which includes the following steps: The algae species used in this experiment were Chlorella sp., purchased from the Seaweed Germplasm Bank of the Institute of Oceanology, Chinese Academy of Sciences. Chlorella sp. was cultivated with an initial inoculum of 0.15 g / L in a 25 cm high, 5 cm diameter, 300 mL conical-bottom column photobioreactor (cone bottom height 5 cm). Under a light intensity of 50 μmol / (m 2 ·s) of continuous fluorescent light, a mixed gas containing 2.5% (v / v) CO2 (the remainder being N2) was introduced into the reactor at a rate of 0.1 vvm to provide a carbon source. The incubation temperature was maintained at 25±1°C. Samples were dried and their dry weight was measured as the biomass concentration of Chlorellasp. The culture medium used for culturing Chlorella sp. is f / 2 culture medium, which is prepared from artificial seawater with a salinity of 35‰.

[0026] The sodium nitrate in this embodiment is added in stages (such as Figure 1 The cumulative concentration of the nitrogen source (NaNO3) in the culture medium was regulated to be 0.2 g / L from day 0 to day 2 (hysteresis phase), 0.65 g / L from day 3 to day 5 (exponential phase), and 0.75 g / L from day 6 to day 8 (stationary phase).

[0027] The final growth of Chlorella sp in this embodiment and Example 1 is as follows Figure 2 As shown in the figure, under the phased nutrient regulation strategy, the maximum biomass concentration of Chlorella sp was measured to be 2.2 g / L. Furthermore, it was observed that Chlorella sp continued to grow, with a trend of increasing biomass concentration. Compared to Example 1, the biomass concentration of Example 1 was lower at the beginning, but exceeded that of Example 1 after the third day and continued to rise, reaching a 54.1% increase on the seventh day. This indicates that regulating the nitrogen source concentration of Chlorella sp during different growth phases can increase the growth rate of photosynthetic microorganisms and the accumulation efficiency of target products.

[0028] Example 2 This embodiment provides a method for coordinating the growth of microorganisms in stages based on light / carbon / nutrients, which includes the following steps: The algae species used in this example were all Chlorella sp., purchased from the Seaweed Germplasm Bank of the Institute of Oceanology, Chinese Academy of Sciences. Chlorella sp. were cultivated with an initial inoculum of 0.15 g / L in a 25 cm high, 5 cm diameter, 300 mL conical-bottom column photobioreactor (5 cm high). A mixed gas containing 2.5% (v / v) CO2 (the remainder being N2) was introduced into the reactor at a rate of 0.1 vvm to provide the carbon source. The incubation temperature was maintained at 25 ± 1°C. Samples were dried and their dry weight was measured as the Chlorella sp. biomass concentration.

[0029] The culture medium used for culturing Chlorella sp. is f / 2 culture medium, which is prepared from artificial seawater with a salinity of 35‰.

[0030] The light intensity in each stage during the culture process of Example 2 (50-100-200-300 μmol / (m 2 ·s)) Figure 3 As shown, specifically, the light intensity during the 0-2 days (hysteresis period) was 50 μmol / (m 2 ·s), and the light intensity on days 2-4 (exponential phase) was 100 μmol / (m 2 ·s), and the light intensity on days 4-6 (exponential phase) was 200 μmol / (m 2 ·s), and the light intensity for the next 6-12 days (stable period) was 300 μmol / (m 2 s); the cumulative concentration of nitrogen source (NaNO3) in the culture medium was regulated to be 0.2 g / L on days 0-2 (lag period), and the cumulative concentration of nitrogen source (NaNO3) in the culture medium was regulated to be 0.75 g / L on days 3-12.

[0031] The growth results of the microalgae Chlorella sp. in this example are as follows Figure 4 As shown, after 11 days of culture, the biomass concentration of Chlorella sp. began to decline. During the entire culture cycle, the maximum biomass concentration of Chlorella sp. in this example under this culture strategy was 3.62 g / L. Under these staged light intensity and nitrogen source concentration conditions, the maximum biomass concentration of Chlorella sp. increased by 82.82% compared to Example 2. In summary, this demonstrates that regulating light intensity and nitrogen source concentration during different microbial growth stages can effectively increase the growth rate of photosynthetic microorganisms and the accumulation efficiency of target products.

[0032] Example 3 This embodiment provides a method for coordinating the growth of microorganisms in stages based on light / carbon / nutrients, which includes the following steps: The algae species used in this example were all Chlorella sp., purchased from the Seaweed Germplasm Bank of the Institute of Oceanology, Chinese Academy of Sciences. Chlorella sp. were cultivated with an initial inoculum of 0.15 g / L in a 300 mL conical-bottom column photobioreactor (5 cm high) measuring 25 cm in height and 5 cm in diameter. The culture temperature was maintained at 25 ± 1°C. Samples were dried and their dry weight was measured as the Chlorella sp. biomass concentration.

[0033] The culture medium used for culturing Chlorella sp. is f / 2 culture medium, which is prepared from artificial seawater with a salinity of 35‰.

[0034] During the cultivation process of this embodiment, the light intensity was adjusted in stages (50-100-200-300 μmol / (m 2 ·s))), such as Figure 5 As shown, specifically, the light intensity during the 0-2 days (hysteresis period) was 50 μmol / (m 2 ·s), and the light intensity on days 2-4 (exponential phase) was 100 μmol / (m 2 ·s), and the light intensity on days 4-6 (exponential phase) was 200 μmol / (m 2 ·s), and the light intensity for the next 6-12 days (stable period) was 300 μmol / (m 2 ·s); sodium nitrate was added in stages, with the nitrogen source (NaNO3) concentration in the culture medium adjusted to 0.2 g / L on days 0-2 (lag phase), and the cumulative nitrogen source (NaNO3) concentration in the culture medium adjusted to 0.75 g / L on days 3-12; the carbon source concentration (CO2%, V / V) was 2.5% on days 0-2 (lag phase), 5% on days 3-6 (exponential phase), and 2.5% on days 7-12 (stable phase).

[0035] The growth results of the microalgae in the cultivation method of this embodiment are as follows: Figure 6As shown, after 11 days of cultivation, the microalgae biomass concentration remained essentially unchanged, indicating that the microalgae culture was in a stable phase. Throughout the entire cultivation cycle, the maximum microalgae biomass concentration was 4.49 g / L. Under the culture conditions of staged light intensity, nitrogen source concentration, and carbon source, the maximum microalgae biomass concentration increased by 126.77% compared to Example 2. Compared to Example 2, Example 3, which added a staged carbon source supply, increased its maximum biomass concentration by 29.56% compared to Example 2. In summary, regulating the light intensity, nitrogen source concentration, and carbon source concentration during different growth phases of microalgae can effectively increase the growth rate of photosynthetic microorganisms and the accumulation efficiency of target products.

[0036] Example 1 This example provides a method for growing microorganisms, which is basically the same as Example 1, with the light intensity and the carbon source concentration remaining unchanged. The difference is that sodium nitrate is added all at once, rather than in stages, specifically: Among them, Example 1 is a light intensity of 50 μmol / (m 2 s ) under continuous fluorescent light illumination, a mixed gas containing 2.5% (v / v) CO2 (the rest was N2) was introduced into the reactor at a rate of 0.1 vvm to provide a carbon source; sodium nitrate was added once at the initial stage of culture at a concentration of 0.2 g / L.

[0037] The growth condition of the microalgae in this example is as follows: after 7 days of cultivation, the microalgae biomass concentration no longer increases and is in a stable growth period of the microalgae, with a maximum biomass concentration of 1.05 g / L.

[0038] Example 2 This example provides a method for growing microorganisms, which is basically the same as Example 2. The concentration of the carbon source introduced remains unchanged. The difference is that the light intensity is maintained constant, and the nitrogen source concentration is added once at the beginning of the culture. Specifically, Among them, Example 2 is a case where the light intensity is 50 μmol / (m 2 ·s) under continuous fluorescent light illumination. The nitrogen source was added once at the beginning of the culture, and the concentration of sodium nitrate was 0.75 g / L. A mixed gas containing 2.5% (v / v) CO2 (the rest was N2) was introduced into the reactor at a rate of 0.1 vvm to provide the carbon source.

[0039] The growth condition of the microalgae in this example is as follows: after 8 days of cultivation, the microalgae biomass concentration no longer increases and is in a stable growth period of the microalgae, with a maximum biomass concentration of 1.98 g / L.

[0040] In summary, the method provided in this application for the coordinated regulation of microbial growth based on light / carbon / nutrients in stages can improve the growth rate of photosynthetic microorganisms and the accumulation efficiency of target products by changing the light intensity parameters, nitrogen source concentration, or carbon source concentration according to the growth requirements of microalgae; and the efficient cultivation of this application should be based on the growth cycle to construct a staged coordinated regulation strategy of multiple factors such as light-carbon-nitrogen-phosphorus to achieve dynamic matching between light intensity, CO2 concentration and nutrient supply, and maximize resource utilization efficiency and product conversion rate.

[0041] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and description of the present invention.

Claims

1. A method for coordinating microbial growth based on light / carbon / nutrients in stages, characterized in that: The method comprises the following steps: cultivating photosynthetic microorganisms by changing control parameters according to the growth cycle of the photosynthetic microorganisms, wherein the control parameters are for dynamically adjusting the concentration of nutrient elements.

2. The method for coordinating microbial growth based on light / carbon / nutrients in stages according to claim 1, characterized in that: The growth cycle of the photosynthetic microorganism includes a hysteresis phase, an exponential phase and a stationary phase.

3. The method for coordinating microbial growth based on light / carbon / nutrients in stages according to claim 1, characterized in that: The control parameters also include dynamically adjusting the light intensity.

4. The method for coordinating microbial growth based on light / carbon / nutrients in stages according to claim 1, characterized in that: The control parameters also include dynamically adjusting the carbon source concentration.

5. The method for coordinating microbial growth based on light / carbon / nutrients in stages according to claim 4, characterized in that: The concentration of the nutrient element is 0.01-0.5 g / L in the hysteresis phase, 0.05-1.5 g / L in the exponential phase, and 0.25-2.0 g / L in the stationary phase.

6. The method for coordinating microbial growth based on light / carbon / nutrients in stages according to claim 1, characterized in that: The concentration of the light intensity during the hysteresis period is 30-150 μmol / m 2 · s, the concentration in the exponential phase is 100-1000 μmol / m 2 ·s, and the concentration in the stable period is 50-500 μmol / m 2 · s.

7. The method for coordinating microbial growth based on light / carbon / nutrients in stages according to claim 1, characterized in that: The carbon source concentration is 1-5% in the hysteresis phase, 5-20% in the exponential phase, and 2-15% in the stationary phase.

8. The method for coordinating microbial growth based on light / carbon / nutrients in stages according to claim 4, characterized in that: The carbon source includes but is not limited to gaseous CO2, and the photosynthetic microorganism includes but is not limited to microalgae.

9. An application of the method for phased coordinated regulation of microbial growth based on light / carbon / nutrients according to any one of claims 1 to 8 in the fields of environmental management, biofuels, medicine and food.

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