A cultivation method for enhancing Spirulina biomass accumulation and its application

Abstract

This invention relates to a cultivation method for enhancing the biomass accumulation of Spirulina and its application. The cultivation method includes the following steps: inoculating Spirulina into a sterile liquid culture medium and culturing it under light conditions of 618-630 nm wavelength, adjusting the light intensity to 120-200 μmol / (m²·s), the photoperiod to 14L:10D-24L:0D, the flash frequency to 30-100 Hz, and the duty cycle to 50%-100%. This invention develops a systematic method for cultivating Spirulina based on light regulation, which is simple to operate and easy to implement. This method can significantly improve the growth rate and biomass accumulation of Spirulina, enabling high-quality large-scale production of Spirulina; it can also improve light energy utilization efficiency and electrical energy utilization efficiency, reducing energy consumption costs.

Description

Technical Field

[0001] This invention belongs to the field of microalgae cultivation technology, and relates to a cultivation method for enhancing the accumulation of Spirulina biomass and its application. Background Technology

[0002] Spirulina is a filamentous cyanobacterium with high nutritional value and wide applications. Its biomass is rich in high-value products such as protein, phycocyanin, and polysaccharides, and has broad application prospects in functional foods, feed additives, and the extraction of bioactive substances. Currently, large-scale cultivation of spirulina mainly uses open raceway ponds or simple photobioreactors, but its growth efficiency is severely limited by the instability of natural light and the low energy efficiency of traditional artificial light sources.

[0003] However, there is currently no systematic light formulation scheme, and in particular, it is difficult to develop a standardized lighting scheme that is replicable, efficient, and energy-saving. This leads to problems such as long growth cycles, low biomass yields, large fluctuations in product quality, and high energy costs in the industrial cultivation of spirulina.

[0004] Therefore, developing a systematic and efficient light formula for spirulina cultivation is of significant technical and economic value for breaking through industry bottlenecks and achieving sustainable, low-cost, high-quality, and large-scale production of spirulina resources. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a cultivation method for improving the accumulation of Spirulina biomass and its application.

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

[0007] In a first aspect, the present invention provides a cultivation method for enhancing the accumulation of Spirulina biomass, the cultivation method comprising the following steps:

[0008] Spirulina was inoculated into sterile liquid culture medium and cultured under light conditions of 618–630 nm wavelength, with the light intensity adjusted to 120–200 μmol / (m²). 2 The light period is 14L:10D to 24L:0D, the flash frequency is 30 to 100 Hz, and the duty cycle is 50% to 100%.

[0009] The specific point values ​​in "618~630 nm" can be selected from 618 nm, 620 nm, 622 nm, 624 nm, 625 nm, 627 nm, 628 nm, 630 nm, etc.

[0010] The light intensity can be specifically selected as 120 μmol / (m²). 2 ·s), 140 μmol / (m2 ·s), 150 μmol / (m 2 ·s), 160 μmol / (m 2 ·s), 170 μmol / (m 2 ·s), 180 μmol / (m 2 ·s), 200 μmol / (m 2 ·s) etc.;

[0011] "14L:10D" refers to "14 hours of light exposure and 10 hours of darkness". The specific methods in "14L:10D~24L:0D" can be 14L:10D, 16L:8D, 18L:6D, 20L:4D, 22L:2D, 24L:0D, etc.

[0012] The flash frequency can be specifically selected as 30 Hz, 40 Hz, 45 Hz, 50 Hz, 55 Hz, 60 Hz, 65 Hz, 70 Hz, 75 Hz, 80 Hz, 90 Hz, 100 Hz, etc.

[0013] The duty cycle can be specifically selected as 50%, 60%, 70%, 80%, 90%, 100%, etc.

[0014] Other specific point values ​​not listed above within the above numerical ranges can be selected, and all are within the protection scope of this invention, and will not be elaborated here.

[0015] This invention develops a systematic method for cultivating spirulina based on light regulation. The method is simple to operate and easy to implement. It can significantly improve the growth rate and biomass accumulation of spirulina, enabling high-quality large-scale production of spirulina. It can also improve light energy utilization efficiency and electrical energy utilization efficiency, and reduce energy consumption costs.

[0016] More preferably, the light intensity is adjusted to 140~160 μmol / (m²). 2 ·s).

[0017] More preferably, the optical period adjustment is 20L:4D to 24L:0D.

[0018] More preferably, the flash frequency is adjusted to 40~60 Hz.

[0019] In this invention, the inoculation amount of Spirulina is 0.05~0.3 g / L based on cell dry weight, for example, 0.05 g / L, 0.1 g / L, 0.15 g / L, 0.2 g / L, 0.23 g / L, 0.25 g / L, 0.28 g / L, 0.3 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.

[0020] In this invention, the culture temperature of the spirulina is 31~35℃, such as 31℃, 31.5℃, 32℃, 32.5℃, 33℃, 33.5℃, 34℃, 34.5℃, 35℃, etc. Other specific values ​​not listed in this range can be selected and are all within the protection scope of this invention, and will not be described in detail here.

[0021] Preferably, the spirulina is kept aerated and 1-5% CO2 is introduced during the cultivation process.

[0022] Preferably, the liquid culture medium is Zarrouk liquid culture medium.

[0023] Secondly, the present invention provides the application of the Spirulina cultivation method according to the first aspect in improving the growth rate and biomass accumulation of Spirulina.

[0024] Thirdly, the present invention provides the application of the Spirulina cultivation method according to the first aspect in improving the light energy utilization efficiency and electrical energy utilization efficiency of Spirulina.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] This invention develops a systematic method for cultivating spirulina based on light regulation. The method is simple to operate and easy to implement. It can significantly improve the growth rate and biomass accumulation of spirulina, enabling high-quality large-scale production of spirulina. It can also improve light energy utilization efficiency and electrical energy utilization efficiency, and reduce energy consumption costs. Detailed Implementation

[0027] 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.

[0028] The Zarrouk liquid culture medium used in the following examples or comparative examples has a formulation of 25.2 g / L. NaHCO3, 2.5 g / L NaNO3, 1.0 g / L NaCl, 0.02 g / L CaCl2·H2O, 1.0 g / L K2SO4, 0.5 g / L K2HPO4, 0.2g / L MgSO4·7H2O, 0.01 g / L FeSO4·7H2O, 0.08 g / L Na2EDTA, 1 ml / L A5. A5 comprises 2.86 g / L. H3BO3, 1.82 g / L MnCl2·4H2O, 0.22 g / L ZnSO4·7H2O, 0.08 g / L CuSO4·5H2O, 0.39 g / L Na2MoO4·2H2O, 0.05 g / L Co3(NO2)·6H2O.

[0029] In the following examples or comparative examples, the dry biomass weight (DCW) (g / L) was determined by gravimetric method. A 0.45 μm pore size glass fiber filter membrane was dried in an oven at 105°C for 2 h to constant weight, cooled in a desiccator, and weighed (m1, g). 50 mL of algal solution was uniformly filtered and washed three times with deionized water to remove salts. The filter membrane carrying algal cells was dried at 105°C for 2 h to constant weight, cooled to 25°C in a desiccator, and weighed (m2, g). The formula for calculating DCW is:

[0030] DCW = (m2 – m1) / 50 × 1000.

[0031] 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:

[0032] μ=[ln(DCW2) – ln(DCW1)] / (t2 – t1);

[0033] DCW2 and DCW1 represent the stem cell weights (g / L) at times t2 and t1, respectively.

[0034] Example 1

[0035] This embodiment provides a method for culturing Spirulina, as detailed below:

[0036] A 1000 mL shake flask / aeration tube system was used, with the temperature controlled at 33℃. An adjustable LED light source was employed, and aeration and 2% CO2 were introduced during the cultivation process to ensure uniform mixing and light exposure of the spirulina.

[0037] Spirulina were inoculated into sterile Zarrouk liquid medium at an inoculation rate of 0.28 g / L (based on cell dry weight) and cultured under light at a wavelength of 620.8 nm with the light intensity adjusted to 160 μmol / (m²). 2 The light period is 24L:0D, the flash frequency is 50 Hz, and the duty cycle is 75%.

[0038] Example 2

[0039] This embodiment provides a method for culturing Spirulina, as detailed below:

[0040] A 1000 mL shake flask / aeration tube system was used, with the temperature controlled at 34℃. An adjustable LED light source was employed, and aeration with 3% CO2 was maintained during the cultivation process to ensure uniform mixing and light exposure of the spirulina.

[0041] Spirulina were inoculated into sterile Zarrouk liquid medium at an inoculation rate of 0.22 g / L (based on cell dry weight) and cultured under 618 nm light intensity at a set concentration of 150 μmol / (m²). 2 The light period is 22L:2D, the flash frequency is 60 Hz, and the duty cycle is 50%.

[0042] Example 3

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

[0044] A 1000 mL shake flask / aeration tube system was used, with the temperature controlled at 32℃. An adjustable LED light source was employed, and aeration with 4% CO2 was maintained during the cultivation process to ensure uniform mixing and light exposure of the spirulina.

[0045] Spirulina were inoculated into sterile Zarrouk liquid medium at a concentration of 0.2 g / L (based on cell dry weight) and cultured under 625 nm light intensity at a set concentration of 140 μmol / (m²). 2 The light period is 20L:4D, the flash frequency is 40 Hz, and the duty cycle is 100%.

[0046] Example 4

[0047] This embodiment provides a method for culturing Spirulina, which differs from Example 1 only in that the light intensity is adjusted to 120 μmol / (m²). 2 ·s), with other conditions remaining unchanged.

[0048] Example 5

[0049] This embodiment provides a method for culturing Spirulina, which differs from Example 1 only in that the light intensity is adjusted to 180 μmol / (m²). 2 ·s), with other conditions remaining unchanged.

[0050] Example 6

[0051] This embodiment provides a method for culturing Spirulina, which differs from Example 1 only in that the light intensity is adjusted to 200 μmol / (m²). 2 ·s), with other conditions remaining unchanged.

[0052] Example 7

[0053] This embodiment provides a method for cultivating Spirulina, which differs from Example 1 only in that the photoperiod is adjusted to 16L:8D, while other conditions remain unchanged.

[0054] Example 8

[0055] This embodiment provides a method for cultivating Spirulina, which differs from Example 1 only in that the photoperiod is adjusted to 14L:10D, while other conditions remain unchanged.

[0056] Example 9

[0057] This embodiment provides a method for cultivating Spirulina, which differs from Embodiment 1 only in that the flash frequency is adjusted to 30 Hz, while other conditions remain unchanged.

[0058] Example 10

[0059] This embodiment provides a method for cultivating Spirulina, which differs from Embodiment 1 only in that the flash frequency is adjusted to 100 Hz, while other conditions remain unchanged.

[0060] Comparative Example 1

[0061] This comparative example provides a method for culturing Spirulina, which differs from Example 1 only in that the light intensity is adjusted to 100 μmol / (m²). 2 ·s), with other conditions remaining unchanged.

[0062] Comparative Example 2

[0063] This comparative example provides a method for culturing Spirulina, which differs from Example 1 only in that the light intensity is adjusted to 250 μmol / (m²). 2 ·s), with other conditions remaining unchanged.

[0064] Comparative Example 3

[0065] This comparative example provides a method for culturing Spirulina, which differs from Example 1 only in that the photoperiod is adjusted to 12L:12D, while other conditions remain unchanged.

[0066] Comparative Example 4

[0067] This comparative example provides a method for cultivating Spirulina, which differs from Example 1 only in that the flash frequency is adjusted to 10 Hz, while other conditions remain unchanged.

[0068] Comparative Example 5

[0069] This comparative example provides a method for cultivating Spirulina, which differs from Example 1 only in that the flash frequency is adjusted to 500 Hz, while other conditions remain unchanged.

[0070] Comparative Example 6

[0071] This comparative example provides a method for culturing Spirulina, which differs from Example 1 only in that the duty cycle is adjusted to 25%, while other conditions remain unchanged.

[0072] Comparative Example 7

[0073] This comparative example provides a method for culturing Spirulina, which differs from Example 1 only in that the culturing is carried out under 633 nm wavelength light conditions, while other conditions remain unchanged.

[0074] Comparative Example 8

[0075] This comparative example provides a method for culturing Spirulina, which differs from Example 1 only in that the culturing is carried out under 652 nm wavelength light conditions, while other conditions remain unchanged.

[0076] Comparative Example 9

[0077] This comparative example provides a method for culturing Spirulina, which differs from Example 1 only in that the culturing is carried out under 473 nm wavelength light conditions, while other conditions remain unchanged.

[0078] Comparative Example 10

[0079] This comparative example provides a method for culturing Spirulina, which differs from Example 1 only in that the culturing is carried out under 469 nm wavelength light conditions, while other conditions remain unchanged.

[0080] Comparative Example 11

[0081] This comparative example provides a method for culturing Spirulina, which differs from Example 1 only in that the temperature during the culturing process is controlled at 36°C, while other conditions remain unchanged.

[0082] Comparative Example 12

[0083] This comparative example provides a method for culturing Spirulina, which differs from Example 1 only in that the temperature during the culturing process is controlled at 30°C, while other conditions remain unchanged.

[0084] Test Example 1

[0085] After culturing for 48 h according to the methods of Examples 1-10 and Comparative Examples 1-12, the dry biomass weight (DCW) and maximum specific growth rate (μmax) of each group were detected, and the results are shown in Table 1.

[0086] Table 1

[0087]

[0088]

[0089] As shown in Table 1, compared with Comparative Examples 1-10, the Spirulina cultivation method of the present invention can significantly improve the growth rate and biomass accumulation of Spirulina through systematic regulation of light wavelength, light intensity, photoperiod, flash frequency and duty cycle.

[0090] Test Example 2

[0091] Comparative experiment between optimized light formulation and conventional white light culture of Spirulina:

[0092] (1) Training setup

[0093] Experimental group: using the optimized light formulation determined in Example 1;

[0094] Control group: Ordinary white light (PPFD 160 μmol / (m)) was used. 2 ·s));

[0095] Both groups were kept in the same conditions during culture: initial inoculation DCW was 0.280 g / L, culture volume was 150 mL, and each group had 3 replicates. The basic experimental parameters are shown in Table 2.

[0096] Table 2

[0097]

[0098] (2) Results:

[0099] After 48 hours, the final dry weight (g / L) of each group was measured. The results showed that the final DCW of the light formulation experimental group was 0.951 ± 0.010 g / L, while that of the control group was 0.721 ± 0.012 g / L. The biomass yield of the experimental group was significantly increased by 31.9% compared with that of the control group (see Table 3).

[0100] Table 3

[0101]

[0102] (3) Optimization of formulation and analysis of white light energy consumption

[0103] Based on the results measured by the integrating sphere spectrometer, the optimal red light formulation and white light parameters were systematically compared, and the results are shown in Table 4.

[0104] Table 4

[0105]

[0106] As shown in Table 4, under the same initial light intensity and culture volume, the photoelectric conversion efficiency of white light was 16.68% higher than that of red light. Even under these conditions, due to the more complete absorption of red light by Spirulina, the light energy utilization efficiency was higher, and the biomass accumulation was still significantly higher than that of the white light group.

[0107] Based on the data results in Table 2, the energy consumption parameters for the two sets were calculated. Detailed parameters are shown in Table 5.

[0108] Table 5

[0109]

[0110] Table 5 shows that the biomass growth rate was 0.671 g / L in the red light group and 0.441 g / L in the white light group. The total power consumption was calculated to be 0.650 kWh for the red light group and 0.553 kWh for the white light group. The energy consumption per unit biomass was 968.94 Wh / g for the red light group and 1253.89 Wh / g for the white light group. The energy consumption per unit biomass for red light was 77.24% of that for white light, resulting in an energy saving of approximately 22.76%.

[0111] Based on the calculation of the light energy utilization rate of microalgae, the total radiant energy of the red light group was 181.99 Wh, and the total radiant energy of the white light group was 247.13 Wh. The light energy utilization rate of red light was 0.00369 g / Wh, and the light energy utilization rate of white light was 0.00178 g / Wh. The energy utilization efficiency of red light radiation was 2.07 times that of white light.

[0112] The above results indicate that although white light lamps have a higher luminous efficiency than pure red light, Spirulina has a greater advantage in absorbing and converting red light, and its biomass accumulation effect is better than that of white light.

[0113] (4) Equivalent photoelectric conversion efficiency

[0114] Based on this, assuming the electro-optical efficiency of red light fixtures remains consistent with that of white light at 44.67%, an equivalent comparison is conducted while maintaining constant radiant power and only improving electro-optical efficiency. The equivalent red light input power can be reduced to 5.658 W, the equivalent total power consumption of red light is 407.38 Wh, and the equivalent energy consumption per unit biomass of red light is 607.12 Wh / g, further enhancing the energy-saving potential.

[0115] This invention provides a complete, efficient, and energy-saving photo-regulated culture scheme for Spirulina through systematic experimental design and verification. The scheme has clearly defined parameters and strong operability, significantly improving the culture efficiency and economics of Spirulina, and has significant industrial application value.

[0116] 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.

[0117] 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.

[0118] 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 cultivation method for enhancing the accumulation of Spirulina biomass, characterized in that, The cultivation method includes the following steps: Spirulina was inoculated into sterile liquid culture medium and cultured under light conditions of 618–630 nm wavelength, with the light intensity adjusted to 120–200 μmol / (m²). 2 The light period is 14L:10D to 24L:0D, the flash frequency is 30 to 100 Hz, and the duty cycle is 50% to 100%.

2. The method for culturing Spirulina according to claim 1, characterized in that, The light intensity is adjusted to 140~160 μmol / (m²). 2 ·s).

3. The method for culturing Spirulina according to claim 1, characterized in that, The optical cycle is adjusted to 20L:4D~24L:0D.

4. The method for culturing Spirulina according to claim 1, characterized in that, The flash frequency is adjusted to 40~60Hz.

5. The method for culturing Spirulina according to any one of claims 1-4, characterized in that, The inoculation amount of Spirulina is 0.05~0.3 g / L based on cell dry weight.

6. The method for culturing Spirulina according to any one of claims 1-5, characterized in that, The culture temperature of the spirulina is 32~34℃.

7. The method for culturing Spirulina according to any one of claims 1-6, characterized in that, During the cultivation of the spirulina, ventilation is maintained and 1-5% CO2 is introduced.

8. The method for culturing Spirulina according to any one of claims 1-7, characterized in that, The liquid culture medium is Zarrouk liquid culture medium.

9. The application of the Spirulina cultivation method according to any one of claims 1-8 in improving the growth rate and biomass accumulation of Spirulina.

10. The application of the Spirulina cultivation method according to any one of claims 1-8 in improving the light energy utilization efficiency and electrical energy utilization efficiency of Spirulina.

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