A method for improving the biomass of chlorella pyrenoidosa by light culture

Abstract

The present application relates to a kind of illumination culture methods for improving chlorella pyrenoidosa biomass, the illumination culture method includes the following steps: chlorella pyrenoidosa stock solution is inoculated in sterile liquid medium, and chlorella pyrenoidosa is illuminated using the composite spectrum of white light and red light.The present application develops a kind of culture method for chlorella pyrenoidosa, i.e.chlorella pyrenoidosa is illuminated using the composite spectrum of white light and red light, the culture method is simple to operate, easy to implement, the method can significantly improve the growth rate and biomass accumulation of chlorella pyrenoidosa, realize the high-quality large-scale production of chlorella pyrenoidosa;It can also reduce the production energy consumption per unit biomass, solves the technical problem that yield increase and consumption reduction are difficult to consider in microalgae culture.

Description

Technical Field

[0001] This invention belongs to the field of microalgae cultivation technology and relates to a light-based cultivation method for increasing the biomass of Chlorella proteoglycans. Background Technology

[0002] Chlorella proteoglycans is a single-celled green algae rich in over 60% high-quality plant protein, 8 essential amino acids, CGF growth factor, chlorophyll, vitamins, and minerals. It has comprehensive nutritional supplementation, immune regulation, detoxification, metabolic enhancement, and cell repair promotion effects. It is widely used in food, health products, aquatic feed, cosmetics, environmental protection, and special medical fields, and is a high-quality biological resource with nutritional, health, ecological, and economic value.

[0003] In the large-scale cultivation of Chlorella proteoglycans, the core contradiction of balancing increased production with reduced energy consumption remains difficult to resolve, becoming a key bottleneck for industrialization. Increased production relies on high density, high light intensity, high aeration, and sufficient nutrients: high density can increase biomass per unit volume, but it exacerbates light shading and reduces deep photosynthetic efficiency, requiring a significant increase in light and aeration energy consumption; high light intensity and high aeration can accelerate photosynthetic carbon fixation, but significantly increase electricity and equipment operating costs; sufficient nitrogen and phosphorus nutrients can ensure rapid proliferation, but increase raw material input and subsequent wastewater treatment costs. On the other hand, reducing energy consumption requires simplifying conditions and reducing energy and nutrient input: weak light, low aeration, and limited nitrogen and phosphorus can reduce costs, but they directly inhibit cell division and biomass accumulation, and may even lead to algal culture collapse due to metabolic imbalance.

[0004] Therefore, developing a cultivation method that can improve the growth rate and biomass accumulation of Chlorella proteoglycans while reducing energy consumption is of significant technical and economic value for breaking through industry bottlenecks and achieving sustainable, low-cost, high-quality, and large-scale production of Chlorella proteoglycans resources. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a light-based culture method for increasing the biomass of Chlorella proteoglycans.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for increasing the biomass of Chlorella proteoglycans under light, the method comprising the following steps:

[0008] Chlorella proteoglycans stock solution was inoculated into sterile liquid culture medium, and the Chlorella proteoglycans were cultured under light using a composite spectrum of white and red light.

[0009] This invention develops a cultivation method for Chlorella proteoglycans, which uses a composite spectrum of white and red light for photoculture. This cultivation method is simple to operate and easy to implement. It can significantly improve the growth rate and biomass accumulation of Chlorella proteoglycans, enabling high-quality large-scale production of Chlorella proteoglycans. It can also reduce the energy consumption per unit biomass, solving the technical problem of balancing increased production and reduced energy consumption in microalgae cultivation.

[0010] Preferably, the wavelength of the red light is 630-660 nm, such as 630 nm, 635 nm, 640 nm, 645 nm, 650 nm, 655 nm, 660 nm, etc. Other specific values ​​not listed in this range can be selected, all of which are within the protection scope of this invention, and will not be described in detail here.

[0011] Preferably, the color temperature of the white light is 4000-4500 K.

[0012] In the light cultivation method involved in this invention, when the wavelength of red light is selected as 630-660 nm and the color temperature of white light is selected as 4000-4500 K, the combined spectrum has a better effect on improving the growth rate and biomass accumulation of Chlorella proteoglycans after illumination.

[0013] Preferably, the ratio of photon flux density of white light to red light in the composite spectrum is 1:2 to 2:1, such as 1:2, 2:3, 1:1, 3:2, 2:1, etc. Other specific values ​​not listed in this range can be selected, all of which are within the protection scope of this invention, and will not be described in detail here.

[0014] In the light cultivation method involved in this invention, when the photon flux density ratio of white light to red light in the composite spectrum is 1:2 to 2:1, it has a better effect on improving the growth rate and biomass accumulation of Chlorella proteoglycans compared to other ratios.

[0015] Preferably, the light-based culture method further includes: adjusting the light intensity to 120-160 μmol / (m²). 2 ·s), for example 120 μmol / (m 2 ·s), 125 μmol / (m 2 ·s), 130 μmol / (m 2 ·s), 135 μmol / (m 2 ·s), 140 μmol / (m 2 ·s), 145 μmol / (m 2 ·s), 150 μmol / (m 2 ·s), 155 μmol / (m 2·s), 160 μmol / (m 2 Other unlisted point values ​​within this range, such as ·s), are all acceptable and fall within the scope of protection of this invention; therefore, they will not be elaborated upon here. More preferably, 145-155 μmol / (m 2 ·s).

[0016] Compared to other light intensities, adjusting the light intensity to 120-160 μmol / (m²) is recommended. 2 •s) is more effective in increasing the growth rate and biomass accumulation of Chlorella proteoglycans.

[0017] Preferably, the light-induced culture method further includes adjusting the photoperiod to 16L:8D-24L:0D, more preferably 22L:2D-24L:0D. Here, "16L:8D" refers to a "16-hour light period and an 8-hour dark period," and the specific settings within "16L:8D-24L:0D" can be 16L:8D, 18L:6D, 20L:4D, 22L:2D, 24L:0D, etc.

[0018] Compared to other photoperiods, adjusting the photoperiod to 16L:8D-24L:0D is more effective in improving the growth rate and biomass accumulation of Chlorella proteoglycans.

[0019] Preferably, the light cultivation method further includes adjusting the flash frequency to 40-60 Hz, such as 40 Hz, 45 Hz, 50 Hz, 55 Hz, 60 Hz, etc. Other specific values ​​not listed within this range can be selected, all of which are within the protection scope of this invention and will not be elaborated here. More preferably, 45-55 Hz.

[0020] Compared to other flash frequencies, adjusting the flash frequency to 40-60 Hz is more effective in improving the growth rate and biomass accumulation of Chlorella proteoglycans.

[0021] Preferably, the light cultivation method further includes: adjusting the light duty cycle to 75%-100%, such as 75%, 80%, 85%, 90%, 95%, 100%, etc. Other specific values ​​not listed within this range can be selected, all of which are within the protection scope of this invention, and will not be described in detail here.

[0022] Preferably, the inoculation amount of the Chlorella proteoglycans stock solution is 0.1-0.2 g / L based on cell dry weight, such as 0.1 g / L, 0.12 g / L, 0.14 g / L, 0.15 g / L, 0.16 g / L, 0.18 g / L, 0.2 g / L, etc. Other specific values ​​not listed within this range can be selected and are all within the protection scope of this invention, and will not be elaborated here.

[0023] Preferably, the culture temperature of the Chlorella proteoglycans is 28-30℃, such as 28℃, 28.5℃, 29℃, 29.5℃, 30℃, etc. Other specific values ​​not listed in this range can be selected, all of which are within the protection scope of this invention, and will not be described in detail here.

[0024] Preferably, the liquid culture medium is BG-11 liquid culture medium.

[0025] Preferably, NaHCO3 is also added to the BG-11 liquid culture medium.

[0026] Preferably, the liquid culture medium is formulated as follows:

[0027] NaHCO3 0.5-1.5 g / L, NaNO3 1-2 g / L, K2HPO4 0.01-0.08 g / L, MgSO4·7H2O 0.01-0.05 g / L, CaCl2·2H2O 0.01-0.05 g / L, Citric acid 0.002-0.01 g / L, Ferric ammonium citrate 0.001-0.01 g / L, Na2EDTA 0.0005-0.0015 g / L, Na2CO3 0.01-0.05 g / L, H3BO3 0.001-0.005 g / L, MnCl2·H2O 0.001-0.003 g / L, ZnSO4·7H2O 0.0001-0.0004 g / L, CuSO4·5H2O 0.00005-0.0001 g / L, Na2MoO4·2H2O 0.0001-0.0005 g / L, Co(NO3)2·6H2O 0.00001-0.0001 g / L; solvent is deionized water.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] This invention develops a cultivation method for Chlorella proteoglycans, which uses a composite spectrum of white and red light for photoculture. This cultivation method is simple to operate and easy to implement. It can significantly improve the growth rate and biomass accumulation of Chlorella proteoglycans, enabling high-quality large-scale production of Chlorella proteoglycans. It can also reduce the energy consumption per unit biomass, solving the technical problem of balancing increased production and reduced energy consumption in microalgae cultivation. Detailed Implementation

[0030] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0031] The formulation of BG-11 liquid culture medium used in the following examples or comparative examples is as follows: NaHCO3 1 g / L, NaNO3 1.50 g / L, K2HPO4 0.04 g / L, MgSO4·7H2O 0.037 g / L, CaCl2·2H2O 0.027 g / L, citric acid 0.006 g / L, ferric ammonium citrate 0.006 g / L, Na2EDTA 0.001 g / L, Na2CO3 0.02 g / L, H3BO3 0.00286 g / L, MnCl2·H2O 0.00181 g / L, ZnSO4·7H2O 0.00022 g / L, CuSO4·5H2O 0.00008 g / L, Na2MoO4·2H2O 0.00039 g / L. g / L, Co(NO3)2·6H2O 0.000041 g / L; solvent is deionized water.

[0032] In the following examples or comparative examples, the final biomass concentration (g / L) was calculated using the filtration-drying-weighing method. The specific procedure was as follows: 10 mL of algal solution was measured, and the filter membrane was pre-dried in a 105°C oven until constant weight. After cooling, the initial mass of the filter membrane (m1, g) was recorded. The algal solution was slowly filtered using a vacuum filtration device to ensure complete retention of algal cells on the filter membrane surface. The algae on the filter membrane were washed twice with deionized water. The filter membrane with attached algal sludge was removed and placed in a 105°C oven for further drying until constant weight. After cooling, the total mass (m2, g) was weighed. The final biomass concentration (DCW, g / L) of the algae was calculated using the following formula:

[0033] DCW = (m2 – m1) / 10 × 1000;

[0034] The maximum specific growth rate (μmax) in the following examples or comparative examples was calculated by monitoring the dynamic changes in biomass, using the following formula:

[0035] μ=[ln(DCW2) – ln(DCW1)] / (t2 – t1);

[0036] DCW2 and DCW1 represent the stem cell weights (g / L) at times t2 and t1, respectively.

[0037] Example 1

[0038] This embodiment provides a method for culturing Chlorella proteoglycans, as detailed below:

[0039] An adjustable spectral light source consisting of an independently controllable 4000K white LED module and a red LED module with a center wavelength of 630 nm is used. Through a precision drive controller, the ratio of photon flux density of white light to red light is adjusted and maintained at 1:1.

[0040] The total illuminance of the aforementioned light source was precisely set and maintained at 150 μmol / (m²). 2 •s); adopts optical periodic (24L:0D) mode; applies pulse modulation with a frequency of 50 Hz and a duty cycle of 100% to the light source through a high-frequency programmable driver.

[0041] Using BG-11 medium as the culture medium, Chlorella proteoglycans were inoculated until the initial stem cell weight was 0.12 g / L. The culture volume was 300 mL / bottle. The bottles were placed in a constant temperature environment at 30℃, and sterile air was continuously introduced at a ventilation rate of 0.5 vvm.

[0042] Example 2

[0043] This embodiment provides a method for culturing Chlorella proteoglycans, as detailed below:

[0044] An adjustable spectral light source consisting of an independently controllable 4300K ​​white LED module and a red LED module with a center wavelength of 650 nm is used. Through a precision drive controller, the ratio of photon flux density of white light to red light is adjusted and maintained at 1:2.

[0045] The total illuminance of the aforementioned light source was precisely set and maintained at 145 μmol / (m²). 2 •s); adopts optical periodicity (22L:2D) mode; applies pulse modulation with a frequency of 55 Hz and a duty cycle of 90% to the light source through a high-frequency programmable driver.

[0046] Using BG-11 medium as the culture medium, Chlorella proteoglycans were inoculated until the initial stem cell weight was 0.12 g / L. The culture volume was 300 mL / bottle. The bottles were placed in a constant temperature environment at 29℃, and sterile air was continuously introduced at a ventilation rate of 0.5 vvm.

[0047] Example 3

[0048] This embodiment provides a method for culturing Chlorella proteoglycans, as detailed below:

[0049] An adjustable spectral light source consisting of an independently controllable 4500K white LED module and a red LED module with a center wavelength of 660 nm is used. Through a precision drive controller, the ratio of photon flux density of white light to red light is adjusted and maintained at 2:1.

[0050] The total illuminance of the aforementioned light source was precisely set and maintained at 155 μmol / (m²). 2 •s); adopts optical periodic (24L:0D) mode; applies pulse modulation with a frequency of 45 Hz and a duty cycle of 75% to the light source through a high-frequency programmable driver.

[0051] Using BG-11 medium as the culture medium, Chlorella proteoglycans were inoculated until the initial stem cell weight was 0.12 g / L. The culture volume was 300 mL / bottle. The bottles were placed in a constant temperature environment at 28℃, and sterile air was continuously introduced at a ventilation rate of 0.5 vvm.

[0052] Example 4

[0053] This embodiment provides a method for culturing Chlorella proteoglycans, which differs from Example 1 only in that the wavelength of the red light is 620 nm, while other conditions remain unchanged.

[0054] Example 5

[0055] This embodiment provides a method for culturing Chlorella proteoglycans, which differs from Example 1 only in that the wavelength of the red light is 700 nm, while other conditions remain unchanged.

[0056] Example 6

[0057] This embodiment provides a method for culturing Chlorella proteoglycans, which differs from Example 1 only in that the color temperature of the white light is 3000 K, while other conditions remain unchanged.

[0058] Example 7

[0059] This embodiment provides a method for culturing Chlorella proteoglycans, which differs from Example 1 only in that the color temperature of the white light is 6000 K, while other conditions remain unchanged.

[0060] Example 8

[0061] This embodiment provides a method for culturing Chlorella proteoglycans, which differs from Example 1 only in that the ratio of photon flux density of white light to red light in the composite spectrum is 1:3, while other conditions remain unchanged.

[0062] Example 9

[0063] This embodiment provides a method for culturing Chlorella proteoglycans, which differs from Example 1 only in that the ratio of photon flux density of white light to red light in the composite spectrum is 3:1, while other conditions remain unchanged.

[0064] Example 10

[0065] This embodiment provides a method for culturing Chlorella proteoglycans, which differs from Example 1 only in that the total light intensity is precisely set and maintained at 120 μmol / (m²). 2 ·s), with other conditions remaining unchanged.

[0066] Example 11

[0067] This embodiment provides a method for culturing Chlorella proteoglycans, which differs from Example 1 only in that the total light intensity is precisely set and maintained at 160 μmol / (m²). 2 ·s), with other conditions remaining unchanged.

[0068] Example 12

[0069] This embodiment provides a method for culturing Chlorella proteoglycans, which differs from Example 1 only in that the total light intensity is precisely set and maintained at 100 μmol / (m²). 2 ·s), with other conditions remaining unchanged.

[0070] Example 13

[0071] This embodiment provides a method for culturing Chlorella proteoglycans, which differs from Example 1 only in that the total light intensity is precisely set and maintained at 180 μmol / (m²). 2 ·s), with other conditions remaining unchanged.

[0072] Example 14

[0073] This embodiment provides a method for culturing Chlorella proteoglycans, which differs from Example 1 only in that it uses a photoperiod (16L:8D) mode, while other conditions remain unchanged.

[0074] Example 15

[0075] This embodiment provides a method for culturing Chlorella proteoglycans, which differs from Example 1 only in that it uses a photoperiod (14L:10D) mode, while other conditions remain unchanged.

[0076] Example 16

[0077] This embodiment provides a method for culturing Chlorella proteoglycans, which differs from Example 1 only in that it uses a photoperiod (12L:12D) mode, while other conditions remain unchanged.

[0078] Example 17

[0079] This embodiment provides a method for culturing Chlorella proteoglycans, which differs from Embodiment 1 only in that: a pulse modulation with a frequency of 40 Hz and a duty cycle of 100% is applied to the light source using a high-frequency programmable driver, while other conditions remain unchanged.

[0080] Example 18

[0081] This embodiment provides a method for culturing Chlorella proteoglycans, which differs from Embodiment 1 only in that: a pulse modulation with a frequency of 60 Hz and a duty cycle of 100% is applied to the light source using a high-frequency programmable driver, while other conditions remain unchanged.

[0082] Example 19

[0083] This embodiment provides a method for culturing Chlorella proteoglycans, which differs from Embodiment 1 only in that: a pulse modulation with a frequency of 10 Hz and a duty cycle of 100% is applied to the light source using a high-frequency programmable driver, while other conditions remain unchanged.

[0084] Example 20

[0085] This embodiment provides a method for culturing Chlorella proteoglycans, which differs from Embodiment 1 only in that: a pulse modulation with a frequency of 70 Hz and a duty cycle of 100% is applied to the light source using a high-frequency programmable driver, while other conditions remain unchanged.

[0086] Comparative Example 1

[0087] This comparative example provides a method for culturing Chlorella proteoglycans, which differs from Example 1 only in that: an adjustable spectral light source consisting of an independently controllable 4000K white LED module and a green LED module with a center wavelength of 525 nm is used. The ratio of photon flux density of white light to red light is adjusted and maintained at 1:1 by a precision drive controller, while other conditions remain unchanged.

[0088] Comparative Example 2

[0089] This comparative example provides a method for culturing Chlorella proteoglycans, which differs from Example 1 only in that: an adjustable spectral light source consisting of an independently controllable 4000K white LED module and a yellow LED module with a center wavelength of 590 nm is used. The ratio of photon flux density of white light to red light is adjusted and maintained at 1:1 by a precision drive controller, while other conditions remain unchanged.

[0090] Comparative Example 3

[0091] This comparative example provides a method for culturing Chlorella proteoglycans, which differs from Example 1 only in that: an adjustable spectral light source consisting of an independently controllable 4000K white LED module and a violet LED module with a center wavelength of 410 nm is used. The ratio of photon flux density of white light to red light is adjusted and maintained at 1:1 by a precision drive controller, while other conditions remain unchanged.

[0092] Comparative Example 4

[0093] This comparative example provides a method for culturing Chlorella proteoglycans, which differs from Example 1 only in that: an adjustable spectral light source consisting of an independently controllable 4000K white LED module and a blue LED module with a center wavelength of 450 nm is used. The ratio of photon flux density of white light to red light is adjusted and maintained at 1:1 by a precision drive controller, while other conditions remain unchanged.

[0094] Comparative Example 5

[0095] This comparative example provides a method for culturing Chlorella proteoglycans, which differs from Example 1 only in that the light source is replaced by a single 4000K white LED module instead of a composite light source. The total light intensity, photoperiod mode, light source application frequency, and duty cycle remain unchanged. All other conditions also remain unchanged.

[0096] Comparative Example 6

[0097] This comparative example provides a method for culturing Chlorella proteoglycans, which differs from Example 1 only in that the light source is replaced by a single red LED module with a center wavelength of 630 nm, while the total light intensity, photoperiod mode, light source application frequency, and duty cycle remain unchanged. Other conditions also remain unchanged.

[0098] Test Example 1

[0099] After culturing *Chlorella proteoglycans* for 120 h according to the methods of Examples 1-20 and Comparative Examples 1-6, the final biomass concentration (g / L) and maximum specific growth rate (μmax, d) of each group were measured. -1 The results of the detection are shown in Table 1.

[0100] Table 1

[0101]

[0102]

[0103] As shown in Table 1, compared with Comparative Examples 1-6, Examples 1-20, namely the Chlorella proteoglycans cultivation method of the present invention, use a composite spectrum of white and red light for light cultivation. The two light sources work together to enhance each other, which can significantly improve the growth rate and biomass accumulation of Chlorella proteoglycans.

[0104] Further comparison of the data results of Examples 1-3 and Examples 4-20 shows that the wavelength of red light, the color temperature of white light, the ratio of photon flux density of white light to red light, light intensity, flash frequency, and photoperiod also affect the growth rate and biomass accumulation of Chlorella proteoglycans to varying degrees.

[0105] Test Example 2

[0106] After culturing *Chlorella proteoglycans* for 120 h according to the methods of Example 1 and Comparative Examples 1-6, the energy consumption per unit biomass (Wh / g) of each group was measured. The calculation formula is as follows:

[0107]

[0108] Where P is the lamp power (W), DCW2 and DCW1 are the final biomass concentration and the initial inoculation biomass concentration (g / L), V is the culture volume, and t is the culture time.

[0109] The results are shown in Table 2.

[0110] Table 2

[0111]

[0112] As shown in Table 2, compared with Comparative Examples 1-6, Example 1, which is the Chlorella proteoglycans cultivation method involved in this invention, uses a composite spectrum of white and red light for illumination cultivation. The two light sources work together to enhance each other, which can significantly reduce the energy consumption per unit biomass of Chlorella proteoglycans.

[0113] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0114] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0115] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for increasing the biomass of Chlorella proteoglycans under light, characterized in that, The light-based culture method includes the following steps: Chlorella proteoglycans stock solution was inoculated into sterile liquid culture medium, and the Chlorella proteoglycans were cultured under light using a composite spectrum of white and red light.

2. The light-based culture method according to claim 1, characterized in that, The wavelength of the red light is 630-660 nm; Preferably, the color temperature of the white light is 4000-4500 K.

3. The light-based culture method according to claim 1 or 2, characterized in that, The ratio of photon flux density of white light to red light in the composite spectrum is 1:2 to 2:

1.

4. The light-based culture method according to any one of claims 1-3, characterized in that, The light-induced culture method further includes: adjusting the light intensity to 120-160 μmol / (m²). 2 ·s), more preferably 145-155 μmol / (m 2 ·s).

5. The light-based culture method according to any one of claims 1-4, characterized in that, The light-induced culture method further includes adjusting the photoperiod to 16L:8D-24L:0D, more preferably 22L:2D-24L:0D.

6. The light-based culture method according to any one of claims 1-5, characterized in that, The light-based cultivation method further includes adjusting the flash frequency to 40-60 Hz, more preferably 45-55 Hz.

7. The light-based culture method according to any one of claims 1-6, characterized in that, The light-based culture method further includes adjusting the light duty cycle to 75%-100%.

8. The light-based culture method according to any one of claims 1-7, characterized in that, The inoculation amount of the Chlorella proteoglycans stock solution is 0.1-0.2 g / L based on cell dry weight.

9. The light-based culture method according to any one of claims 1-8, characterized in that, The culture temperature of the Chlorella proteoglycans is 28-30℃.

10. The light-based culture method according to any one of claims 1-9, characterized in that, The liquid culture medium is BG-11 liquid culture medium.

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