A method for efficient recovery of haematococcus cells using organic flocculation

Abstract

The application belongs to the field of algal resource utilization and bioengineering technology, and specifically discloses a method for collecting Haematococcus pluvialis cells by using organic flocculation, and the specific steps are as follows: after Haematococcus pluvialis is completely converted into red spores, cassava starch is first added into the algal liquid, 80 mg of cassava starch is added into each liter of the algal liquid, uniform stirring is performed, and standing is performed for 2 hours; then, sodium alginate / chitosan composite flocculant is added into the algal liquid, 40 mg of the sodium alginate / chitosan composite flocculant is added into each liter of the algal liquid, uniform stirring is performed, standing is performed for 1 hour, the precipitate is collected, and Haematococcus pluvialis cells are obtained. The cassava starch and the sodium alginate / chitosan composite flocculant used in the application can significantly improve the flocculation rate of Haematococcus pluvialis, are non-toxic, environmentally friendly, easy to degrade, and low in cost; the method is simple to operate, difficult to apply in industrialization on a large scale, and has a wide market application prospect.

Description

Technical Field

[0001] This invention relates to the fields of algal resource utilization and bioengineering technology, specifically to a method for harvesting Haematococcus pluvialis cells using organic flocculation. Background Technology

[0002] Haematococcus lacustris is a freshwater single-celled green algae and is currently the species with the highest known natural astaxanthin content. Astaxanthin, due to its excellent coloring effect and antioxidant capacity, is widely used in the food, cosmetics, pharmaceutical, and biological feed industries, becoming a research hotspot for high-value-added components among economically important microalgae, with broad development prospects and utilization value. However, due to the small size of Haematococcus lacustris cells and the similarity between cell density and substrate culture medium density, the harvesting process is difficult and energy-intensive, becoming one of the technical challenges for its large-scale industrialization. Therefore, developing efficient and environmentally friendly harvesting methods to improve the harvest rate of Haematococcus lacustris is beneficial to promoting its development and application.

[0003] Currently, the main harvesting methods for Haematococcus pluvialis include sedimentation, centrifugation, and flocculation. In actual production, the cultivation of Haematococcus pluvialis generally involves two stages: growth and reproduction, and astaxanthin accumulation. After the astaxanthin accumulation stage, the algal cells completely transform into red spores. At this point, mature Haematococcus pluvialis cells can be harvested through sedimentation and centrifugation concentration. However, while centrifugation is fast and reliable, it incurs high energy consumption and costs during operation. Furthermore, the high rotation speed can cause the algal cells to rupture due to friction against the centrifuge wall, leading to the dissolution of nutrients such as astaxanthin. Flocculation, on the other hand, is low-cost and easy to operate, making it suitable for large-scale harvesting and considered a more economical and reliable method.

[0004] Based on their raw materials, flocculants can be classified into inorganic flocculants, organic flocculants, and microbial flocculants. Inorganic flocculants are inexpensive, easy to use, and have mature production processes, but they have the disadvantage of potentially polluting water bodies with residual metal ions. While microbial flocculants are biodegradable, pollution-free, and have good flocculation effects, their high cost makes them unsuitable for large-scale production. Therefore, organic flocculants have advantages overall. However, their application in the harvesting of *Hylocereus undatus* is currently limited. Most applications use a single organic flocculant, such as chitosan, resulting in low recovery rates. There are also applications that combine organic and inorganic flocculants, such as the flocculant and its uses disclosed in CN104342374B, which describes a method for treating microalgae culture media. This flocculant contains chitosan, a water-soluble organic acid, and a water-soluble metal compound. The water-soluble organic acid is at least one of acetic acid, benzoic acid, lactic acid, chloroacetic acid, propionic acid, formic acid, oxalic acid, citric acid, and tartaric acid. The water-soluble metal compound is at least one of aluminum chloride, ferric chloride, aluminum sulfate, potassium aluminum sulfate, ferric sulfate, ferrous chloride, and ferrous sulfate. This flocculant is suitable for flocculation treatment of microalgae culture media containing at least one of the genera *Chlorella*, *Botrytis*, *Micrococcus*, *Hylocereus*, and *Chlorella*. While it effectively improves the recovery rate of microalgae, the use of water-soluble metal compounds inevitably leads to contamination. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for effectively harvesting Haematococcus pluvialis cells using organic flocculation, which features high flocculation rate, non-toxicity, environmental friendliness, low cost, and simple operation.

[0006] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows:

[0007] A method for effectively harvesting Haematococcus pluvialis cells using organic flocculation involves adding cassava starch and sodium alginate / chitosan composite flocculant to the Haematococcus pluvialis algal solution, allowing flocculation and precipitation, and then separating the solid and liquid to obtain the Haematococcus pluvialis cells.

[0008] This invention utilizes three organic flocculants: cassava starch, sodium alginate, and chitosan. All three are natural organic compounds, making them more environmentally friendly than inorganic flocculants and preventing pollution. Compared to synthetic organic flocculants, they significantly reduce costs and are easily degradable. More specifically, cassava starch exhibits better flocculation than other starch flocculants; the combination of sodium alginate and chitosan provides better flocculation and stability than using them separately; and the combined flocculant of cassava starch and sodium alginate / chitosan achieves better flocculation of Haematococcus pluvialis cells than either cassava starch alone or the sodium alginate / chitosan alone.

[0009] Preferably, the specific steps of the method are as follows: First, add cassava starch to the Haematococcus pluvialis algal solution, stir evenly, and let it stand; then add sodium alginate / chitosan composite flocculant, stir evenly, let it stand, collect the precipitate, and obtain Haematococcus pluvialis cells. Adding the organic flocculant in two steps, with cassava starch added first and sodium alginate / chitosan composite flocculant added later, results in better flocculation. The reason is as follows: Starch molecules contain a large number of active groups, which can effectively capture algal cells to form flocs and promote sedimentation. However, considering that the size of the formed flocs is small and affects the sedimentation rate, and that starch residue in the water can easily cause organic pollution, sodium alginate / chitosan composite flocculant is added afterward. This flocculant can not only flocculate algal cells, but also perform secondary flocculation of unsettled starch-algal cell flocs and unattached starch molecules, effectively improving the flocculation rate of Haematococcus pluvialis.

[0010] Preferably, 60-100 mg of cassava starch is added per liter of algae solution, and the standing time is 1-3 hours.

[0011] Furthermore, an average of 80 mg of cassava starch was added per liter of algae solution, and the solution was allowed to stand for 2 hours.

[0012] Preferably, the sodium alginate / chitosan composite flocculant is prepared by combining sodium alginate and chitosan in a mass ratio of (1-3):1; an average of 20-60 mg of sodium alginate / chitosan composite flocculant is added per liter of algal solution, and the standing time is 30-90 min.

[0013] Furthermore, the sodium alginate / chitosan composite flocculant is prepared by combining sodium alginate and chitosan in a mass ratio of 1:1; an average of 40 mg of sodium alginate / chitosan composite flocculant is added per liter of algal solution, and the settling time is 60 min.

[0014] Furthermore, the preparation process of the sodium alginate / chitosan composite flocculant is as follows: a 1% sodium alginate solution is prepared using distilled water as a solvent; a 1% chitosan solution is prepared using 1% acetic acid as a solvent; the chitosan solution and sodium alginate solution are allowed to stand overnight, then mixed in equal volumes and stirred continuously, then acetone is added to precipitate the precipitate, the precipitate is washed with distilled water and freeze-dried to obtain the sodium alginate / chitosan composite flocculant.

[0015] Preferably, the cassava starch is added after the Haematococcus pluvialis has completely transformed into red spores, because at this time astaxanthin has completed its accumulation, and the algal cells are of higher value. Specifically, this can be observed with the naked eye when the algal solution has turned noticeably red, or observed under a microscope when most of the algal cells have turned red.

[0016] The present invention has the following beneficial effects:

[0017] 1. This invention uses cassava starch and sodium alginate / chitosan composite flocculant to flocculate Haematococcus pluvialis algal liquid, which significantly improves the flocculation rate of Haematococcus pluvialis, with the highest flocculation rate reaching 91.4%, laying a good technical foundation for the commercial application of Haematococcus pluvialis.

[0018] 2. The cassava starch, sodium alginate, and chitosan used in this invention are all natural organic materials, which are non-toxic, environmentally friendly, easy to degrade, and low in cost. They have significant advantages compared with inorganic flocculants and artificially synthesized organic flocculants.

[0019] 3. The method proposed in this invention is simple to operate, requires no complex parameter control, is easy to apply on a large scale in industrial applications, and has broad market application prospects. Detailed Implementation

[0020] The present invention will be further described below with reference to specific embodiments.

[0021] Example 1

[0022] The effects of different types of starch flocculants on the flocculation rate of Haematococcus pluvialis were compared. 100 mL of Haematococcus pluvialis spores (completely converted to red spores) was placed in a 150 mL Erlenmeyer flask, and the initial absorbance A0 was measured at 680 nm. Equal amounts (5.0 mg) of three different starch flocculants (corn starch, potato starch, and cassava starch) were added, stirred until dissolved, and allowed to stand at room temperature for the same time (1 h). The absorbance A of the supernatant was then measured, and the flocculation rate of Haematococcus pluvialis was calculated using the formula: W = (A0 - A) / A0 × 100%.

[0023] The results are shown in Table 1. As can be seen from Table 1, cassava starch has the best flocculation effect on Haematococcus pluvialis as a flocculant, with a flocculation rate of 80.3%.

[0024] Table 1. Effects of different types of starch flocculants on the flocculation rate of Haematococcus pluvialis.

[0025]

[0026] Example 2

[0027] The effects of different amounts of cassava starch flocculant on the flocculation rate of Haematococcus pluvialis at different time points were compared. 100 mL of Haematococcus pluvialis algal solution (completely converted into red spores) was taken into a 150 mL Erlenmeyer flask, and the initial absorbance A0 was measured at 680 nm. 2.0 mg, 4.0 mg, 6.0 mg, 8.0 mg, and 10.0 mg of cassava starch flocculant were added to the algal solution, stirred until dissolved, and allowed to stand at room temperature. The absorbance A of the supernatant was measured after 1, 2, and 3 hours, and the flocculation rate of Haematococcus pluvialis was calculated (calculation method same as in Example 1).

[0028] The results are shown in Table 2. As can be seen from Table 2, considering both time and cost, cassava starch content of 8.0 mg (concentration of 80 mg / L) and flocculation time of 2 h have the best flocculation effect on Haematococcus pluvialis, with a flocculation rate of 87.1%.

[0029] Table 2. Effects of different cassava starch flocculants on the flocculation rate of Haematococcus pluvialis at different time points.

[0030]

[0031] Example 3

[0032] The effects of different proportions of composite flocculants on the flocculation rate of Haematococcus pluvialis were compared. 100 mL of Haematococcus pluvialis solution treated with 8.0 mg cassava starch (concentration 80 mg / L) and flocculation time of 2 h in Example 2 was placed in a 150 mL Erlenmeyer flask, and the initial absorbance A0 was measured at 680 nm. Equal amounts (3.0 mg) of sodium alginate / chitosan composite flocculants in different proportions (mass ratios of 1:3, 1:1, and 3:1) were added, stirred until dissolved, and allowed to stand at room temperature for the same time (60 min). The absorbance A of the supernatant was then measured, and the flocculation rate of Haematococcus pluvialis was calculated. The preparation method of sodium alginate / chitosan composite flocculant is as follows: Sodium alginate solutions with a mass fraction of 1% and 3% are prepared using distilled water as a solvent, and chitosan solutions with a mass fraction of 1% and 3% are prepared using acetic acid with a mass fraction of 1%. The solutions are left to stand overnight. Then, equal volumes of 1% sodium alginate solution and 3% chitosan solution, 1% sodium alginate solution and 1% chitosan solution, and 3% sodium alginate solution and 1% chitosan solution are mixed and stirred continuously. Acetone is then added to precipitate the product. The precipitate is washed with distilled water and freeze-dried to obtain the sodium alginate / chitosan composite flocculant with the desired proportion.

[0033] The results are shown in Table 3. As can be seen from Table 3, the sodium alginate: chitosan = 1:1 composite addition has the best flocculation effect on Haematococcus pluvialis, with a flocculation rate of 90.5%.

[0034] Table 3. Effects of different proportions of composite flocculants on the flocculation rate of Haematococcus pluvialis.

[0035]

[0036] Example 4

[0037] The effects of different concentrations of composite flocculants and different flocculation times on the flocculation rate of Haematococcus pluvialis were compared. 100 mL of Haematococcus pluvialis algal solution treated with 8.0 mg cassava starch (concentration 80 mg / L) and flocculation time of 2 h (as in Example 2) was placed in a 150 mL Erlenmeyer flask, and the initial absorbance A0 was measured. 2.0 mg, 4.0 mg, and 6.0 mg of sodium alginate / chitosan composite flocculant (1:1) were added to the algal solution, stirred until dissolved, and allowed to stand at room temperature. The supernatant was collected after 30, 60, and 90 min, and the absorbance A was measured to calculate the flocculation rate of Haematococcus pluvialis.

[0038] The results are shown in Table 4. As can be seen from Table 4, considering both time and cost, the sodium alginate / chitosan composite flocculant with a content of 4.0 mg (concentration of 40 mg / L) and a flocculation time of 60 min had the best flocculation effect on Haematococcus pluvialis, with a flocculation rate of 91.4%.

[0039] Table 4. Effects of different contents of composite flocculant on the flocculation rate of Haematococcus pluvialis at different times.

[0040]

[0041] In summary, the optimal method for harvesting Haematococcus pluvialis cells selected by this invention is as follows: After Haematococcus pluvialis has completely transformed into red spores, cassava starch is first added to the algal solution at a concentration of 80 mg per liter of algal solution, stirred evenly, and allowed to stand for 2 hours; then sodium alginate / chitosan composite flocculant is added at a concentration of 40 mg per liter of algal solution, stirred evenly, allowed to stand for 1 hour, and then the precipitate is collected by filtration to obtain Haematococcus pluvialis cells.

[0042] Example 5

[0043] Compared to the optimal solution described above, this embodiment changed the order of adding cassava starch and sodium alginate / chitosan composite flocculant, achieving a flocculation rate of 90.7%. It is evident that changing the order of flocculant addition slightly reduced the flocculation effect, but it still exhibits significant advantages compared to existing conventional methods for harvesting Haematococcus pluvialis cells.

[0044] Example 6

[0045] Compared to the optimal solution described above, this embodiment adds cassava starch and sodium alginate / chitosan composite flocculant simultaneously, with the same total settling time, achieving a flocculation rate of 90.9%. It is evident that while the flocculation effect decreases after simultaneous addition, it still exhibits significant advantages compared to existing conventional methods for harvesting Haematococcus pluvialis cells.

[0046] Comparative Example 1

[0047] This comparative example uses the existing conventional method for harvesting Haematococcus pluvialis cells, specifically as follows: 30 mg / L chitosan was added to the Haematococcus pluvialis solution, the flocculation time was 120 min, and the flocculation rate was 74.8%.

[0048] Comparative Example 2

[0049] Compared with the above optimal solution, this comparative example only added cassava starch and tested different times and different amounts of addition, with a maximum flocculation rate of 87.1%.

[0050] Comparative Example 3

[0051] Compared with the above optimal solution, this comparative example only added sodium alginate / chitosan composite flocculant and tested different times and different addition amounts, with a maximum flocculation rate of 88.7%.

[0052] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. Any changes made by those skilled in the art after reading the specification of the present invention, as long as they are within the scope of the claims of the present invention, will be protected by patent law.

Claims

1. A method for effectively harvesting Haematococcus pluvialis cells using organic flocculation, characterized in that: The process involves adding cassava starch and sodium alginate / chitosan composite flocculant to Haematococcus pluvialis liquid, causing flocculation and precipitation, followed by solid-liquid separation to obtain Haematococcus pluvialis cells.

2. The method for effectively harvesting Haematococcus pluvialis cells using organic flocculation according to claim 1, characterized in that: The specific steps are as follows: First, add cassava starch to the Haematococcus pluvialis algal solution, stir evenly and let stand; then add sodium alginate / chitosan composite flocculant, stir evenly and let stand, collect the precipitate, and obtain Haematococcus pluvialis cells.

3. The method for effectively harvesting Haematococcus pluvialis cells using organic flocculation according to claim 2, characterized in that: Add 60-100 mg of cassava starch per liter of algae solution and let it stand for 1-3 hours.

4. The method for effectively harvesting Haematococcus pluvialis cells using organic flocculation according to claim 3, characterized in that: On average, 80 mg of tapioca starch was added per liter of algae solution, and the solution was allowed to stand for 2 hours.

5. The method for effectively harvesting Haematococcus pluvialis cells using organic flocculation according to any one of claims 2-4, characterized in that: The sodium alginate / chitosan composite flocculant is prepared by combining sodium alginate and chitosan in a mass ratio of (1-3):1; an average of 20-60 mg of sodium alginate / chitosan composite flocculant is added per liter of algal solution, and the standing time is 30-90 min.

6. The method for effectively harvesting Haematococcus pluvialis cells using organic flocculation according to claim 5, characterized in that: The sodium alginate / chitosan composite flocculant is prepared by combining sodium alginate and chitosan in a mass ratio of 1:1; an average of 40 mg of sodium alginate / chitosan composite flocculant is added per liter of algal solution, and the settling time is 60 min.

7. The method for effectively harvesting Haematococcus pluvialis cells using organic flocculation according to claim 6, characterized in that: The preparation process of the sodium alginate / chitosan composite flocculant is as follows: a 1% sodium alginate solution is prepared using distilled water as a solvent; a 1% chitosan solution is prepared using 1% acetic acid as a solvent; the chitosan solution and sodium alginate solution are left to stand overnight, then mixed in equal volumes and stirred continuously, and acetone is added to precipitate the precipitate. The precipitate is washed with distilled water and freeze-dried to obtain the sodium alginate / chitosan composite flocculant.

8. The method for effectively harvesting Haematococcus pluvialis cells using organic flocculation according to claim 1, characterized in that: The cassava starch was added after the Haematococcus pluvialis had completely converted into red spores.

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