Method for harvesting bait microalgae and application thereof

Abstract

The invention relates to the field of microalgae organisms, and discloses a method for harvesting bait microalgae, which is characterized by comprising the following steps: (1) mixing diatomite and chitosan with a bait microalgae culture solution for flocculation; and (2) introducing a ferric ion compound into the mixed solution for sedimentation. The method for harvesting the bait microalgae, disclosed by the invention, is suitable for various bait microalgae and bait microalgae in different culture periods; according to the method, the applicable initial algae liquid concentration is high, the applicable initial algae liquid concentration range is large, the harvesting time is short, and the flocculation efficiency is high.

Description

Technical Field

[0001] The present invention relates to the field of microalgae biology, and particularly relates to a method for harvesting bait microalgae and its application. Background Art

[0002] Bait algae are micro unicellular algae used as breeding baits for precious marine cultured animals such as prawns, zoea larvae of Scylla serrata, scallops, and sea cucumbers. Bait microalgae play an irreplaceable role in artificial aquaculture breeding. It is not only the direct initial food for shellfish, prawn larvae, and some fish larvae, but also the necessary food for secondary bait organisms in freshwater and seawater zooplankton aquaculture. However, due to the small cell volume and low density of microalgae, harvesting is difficult and costly.

[0003] Currently, among microalgae harvesting methods, chemical flocculation is one of the most widely used technologies in microalgae harvesting. The surfaces of microalgae cells are mostly negatively charged, so the chemical flocculants used are cationic flocculants, including metal salts and polymer compounds. Commonly used metal ions for chemical flocculation are A1 3+ 、Fe 3+ 、Zn 2+ etc. These metal ions can strongly adsorb on the surfaces of microalgae cells, electrically neutralize the negative charges on their surfaces, eliminate the electrostatic repulsion between cells, and thus achieve the flocculation effect. Chemical flocculation has the advantages of simple and convenient operation and no energy requirement. However, if the dosage of the chemical flocculant is too large, there will be a large amount of metal ions in the obtained microalgae biomass, which is likely to affect the subsequent development and utilization of microalgae.

[0004] "Optimization of Chlorella vulgaris Harvesting by Alum Flocculation Using Response Surface Methodology" (Genomics and Applied Biology, 2021(007):040.) discloses a process for harvesting Chlorella vulgaris using alum as a flocculant. Under the conditions of an alum concentration of 1.2 g / L, an initial OD 680 value of 0.8, and a flocculation time of 30 min, the flocculation efficiency of Chlorella vulgaris can reach 98.49%. However, the initial OD 680 value of the Chlorella vulgaris solution is only 0.8. As the concentration of the Chlorella vulgaris solution increases, the flocculation efficiency decreases sharply. When the initial OD 680 value is 2, the flocculation efficiency is only 65%.

[0005] "Flocculation of Isochrysis galbana by Three Flocculants" (Chinese Journal of Ecology, 2012, 31(10): 2631-2634) studied the flocculation efficiency of three flocculants on algal cells, the effect on the total lipid content of algal bodies, and the morphological changes of algal cells. The results showed that the precipitation rate of ferric chloride was relatively fast, and the precipitation process was basically completed in the 2nd hour after addition; the precipitation of alum lasted for a longer time, up to 12 h; the flocculation effect of chitosan was the worst, and there was still no obvious sedimentation of algal bodies after 16 h of flocculation, so it was not suitable for the flocculation of this alga. Moreover, when the concentration of ferric chloride ≥ 20 mg / L and the concentration of alum ≥ 80 mg / L, more than 90% of Isochrysis galbana (OD 430 was about 0.4) could be flocculated and precipitated. However, the precipitation time of this method was too long, and the initial concentration of algal solution was relatively low.

[0006] Different species of microalgae and different culture periods of the same microalgae have different amounts of electric charge on the surface of microalgal cells. Therefore, the current methods for harvesting bait microalgae are often only applicable to microalgae in a certain culture period and only applicable to a single species of microalgae, such as only applicable to Chlorella or only applicable to Isochrysis galbana. Moreover, in order to be applied to large-scale aquaculture, the initial concentration of bait microalgae used in the harvesting method cannot be too low. Summary of the Invention

[0007] The purpose of the present invention is to overcome the problems existing in the prior art that the methods for harvesting bait microalgae cannot be applied to multiple bait microalgae, cannot be applied to bait microalgae in multiple culture periods, and have too long sedimentation time, and to provide a method for harvesting bait microalgae and its application.

[0008] To achieve the above purpose, in the first aspect of the present invention, a method for harvesting bait microalgae is provided, wherein the method includes:

[0009] (1) Mix diatomite and chitosan with the bait microalgae culture solution for flocculation;

[0010] (2) Introduce a trivalent iron ion compound into the mixed solution for sedimentation.

[0011] In the second aspect of the present invention, an application of the method described in the first aspect of the present invention in aquaculture is provided.

[0012] The method for harvesting bait microalgae described in the present invention is applicable to multiple bait microalgae and bait microalgae in different culture periods; the method of the present invention is applicable to a high initial concentration of algal solution, has a large applicable range of initial algal solution concentration, short harvesting time, and high flocculation efficiency. Moreover, the method has simple operation and low cost.

[0013] Compared with the conventional methods for harvesting algal cells in the art, at the same algal cell concentration, the concentration of the ferric ion compound used in the method of the present invention is lower, reducing the impact of introducing excessive metal ions on the use of microalgae. Moreover, the flocculation efficiency of the harvesting method is high, and the nutritional value of the harvested microalgae is not easily damaged. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 shows the influence of the initial algal liquid concentration on the flocculation efficiency of Isochrysis galbana.

[0015] Figure 2 shows the influence of the cultivation period of the bait microalgae on the flocculation efficiency of Isochrysis galbana.

[0016] Figure 3 is the influence of the concentration of FeCl 3 on the flocculation efficiency of Isochrysis galbana.

[0017] Figure 4 shows the influence of the composition of the diatomite and chitosan mixture on the flocculation efficiency of Isochrysis galbana.

[0018] Figure 5 shows the influence of the concentration of the diatomite and chitosan mixture on the flocculation efficiency of Isochrysis galbana.

[0019] Figure 6 shows the influence of the addition sequence and the composition of the flocculant on the flocculation effect.

[0020] Figure 7 shows the flocculation effects of different bait microalgae. DETAILED DESCRIPTION OF THE INVENTION

[0021] The endpoints and any values disclosed herein are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0022] In the present invention, the final concentration refers to the concentration in the mixed system, and the mixed system refers to a system containing the bait microalgae culture solution and other substances added externally for harvesting microalgae, such as diatomite, chitosan, ferric ion compounds, etc.

[0023] The first aspect of the present invention provides a method for harvesting bait microalgae, wherein the method includes:

[0024] (1) Mixing diatomite and chitosan with the bait microalgae culture solution for flocculation;

[0025] (2) Introduce a ferric ion compound into the mixed solution for sedimentation.

[0026] The inventors of the present invention unexpectedly found during the experiment that, compared with adding only ferric ion compounds, the flocculation efficiency can be greatly improved by adding diatomaceous earth, chitosan, and ferric ion compounds for harvesting; moreover, the addition sequence of diatomaceous earth, chitosan, and ferric ion compounds affects the flocculation efficiency. If ferric ion compounds are added first and then diatomaceous earth and chitosan, the flocculation efficiency is not much different from that of adding only ferric ion compounds; however, if a mixture of diatomaceous earth and chitosan is added first and then ferric ion compounds, the flocculation efficiency is improved.

[0027] In the field of microalgae harvesting, when using flocculants for harvesting according to the reports in the literature and the conventional methods in this field, even considering the harvesting efficiency and cost, the sedimentation time is still several hours or days, and the entire harvesting time is relatively long. Moreover, the inventors of the present invention found in the research that if the sedimentation time is too long, the flocculation efficiency will decrease. By using the harvesting method described in the present invention, the sedimentation time can be controlled within one hour while maintaining a high flocculation efficiency. In the present invention, preferably, the sedimentation time is 5 - 40 min, for example, it can be 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, and any range composed of any two of these values. More preferably, the sedimentation time is 10 - 30 min, and further preferably 15 - 25 min.

[0028] In the present invention, in order to make the diatomaceous earth, chitosan, and the bait microalgae culture solution mix evenly, and further improve the flocculation efficiency and shorten the sedimentation time, preferably, stirring is carried out during mixing, and the stirring time only needs to make the diatomaceous earth, chitosan, and the bait microalgae culture solution mix evenly. For example, it can be 5 - 20 s.

[0029] In the present invention, in order to make the ferric ion compound mix evenly with the diatomaceous earth, chitosan, and the bait microalgae culture solution, and further improve the flocculation efficiency and shorten the sedimentation time, preferably, stirring is carried out after introducing the ferric ion compound and then sedimentation, and the stirring time can be 3 - 10 min.

[0030] In the present invention, the temperature of the sedimentation can be above 0°C. In order to improve the flocculation effect, preferably, the temperature of the sedimentation is 15°C - 50°C, and more preferably 20 - 25°C.

[0031] Microalgae at different cultivation stages have different surface charges. Therefore, in the field of microalgae harvesting, different harvesting methods are usually applicable to microalgae at different cultivation stages. However, the inventors of the present invention found that, using the harvesting method of the present invention, in the culture solution of the bait microalgae, the cultivation stage of the bait microalgae can be any cultivation stage, for example, it can be the lag phase, the early logarithmic phase, the mid-logarithmic phase, the late logarithmic phase or the stationary phase. Although in some preferred embodiments of the present invention, the bait microalgae in the late logarithmic phase are selected, those skilled in the art should understand that the cultivation stage of the bait microalgae is not limited to the late logarithmic phase.

[0032] In the present invention, the OD value refers to the absorbance (optical density, OD) value of the microalgae solution at the maximum absorption wavelength of the microalgae.

[0033] In the present invention, the initial OD value of the culture solution of the bait microalgae is not particularly limited. For example, it can be 10 or less, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, and any range composed of any two of these values. Preferably, the initial OD value of the culture solution of the bait microalgae is 5 or less.

[0034] In the field of microalgae harvesting, the microalgae harvesting method that can be used in large-scale aquaculture, that is, the commercial microalgae harvesting method, the maximum initial OD value of the culture solution of the applicable bait microalgae is 2. In the present invention, for commercial considerations, in some preferred embodiments, the initial OD of the culture solution of the bait microalgae 680 is between 0.5 and 3, more preferably between 0.5 and 2.5.

[0035] In the present invention, the type of the bait microalgae is not particularly limited, and it is a conventional bait microalgae that can be used in aquaculture. For example, it can be microalgae of Bacillariophyta, Chrysophyta and Chlorophyta, such as at least one of Tetraselmis, Phaeodactylum, Nitzschia, Chaetoceros, Skeletonema, Isochrysis, Heterogloea, Nannochloropsis, Chlorella.

[0036] In the present invention, preferably, the bait microalgae are at least one of Tetraselmis, Phaeodactylum, Nannochloropsis, Isochrysis, Chlorella.

[0037] In the present invention, preferably, the Tetraselmis is at least one of Tetraselmis subcordiformis, Tetraselmis pyriformis and Tetraselmis helgolandica.

[0038] In the present invention, preferably, the Phaeodactylum is Phaeodactylum tricornutum.

[0039] In the present invention, preferably, the Nannochloropsis is at least one of Nannochloropsis oceanica, Nannochloropsis oculata, Nannochloropsis limnetica, Nannochloropsis gaditana, Nannochloropsis salina and Nannochloropsis granulata.

[0040] In the present invention, preferably, the Isochrysis is at least one of Isochrysis galbana, Dicrateria inornata and Isochrysis zhanjiangensis.

[0041] In the present invention, preferably, the Chlorella is at least one of Chlorella pyrenoidosa, Chlorella vulgaris, Chlorella ellipsoidea, Chlorella sorokiniana and Chlorella zofingiensis.

[0042] In some preferred embodiments of the present invention, the bait microalgae is at least one of Tetraselmis subcordiformis, Phaeodactylum tricornutum, Nannochloropsis oceanica, Isochrysis galbana and Chlorella pyrenoidosa.

[0043] In the present invention, the final concentration of diatomite in the mixed system can be 100 - 500 mg / L, such as 100 mg / L, 110 mg / L, 120 mg / L, 130 mg / L, 140 mg / L, 150 mg / L, 160 mg / L, 170 mg / L, 180 mg / L, 190 mg / L, 200 mg / L, 210 mg / L, 220 mg / L, 230 mg / L, 240 mg / L, 250 mg / L, 260 mg / L, 270 mg / L, 280 mg / L, 290 mg / L, 300 mg / L, 310 mg / L, 320 mg / L, 330 mg / L, 340 mg / L, 350 mg / L, 360 mg / L, 370 mg / L, 380 mg / L, 390 mg / L, 400 mg / L, 410 mg / L, 420 mg / L, 430 mg / L, 440 mg / L, 450 mg / L, 460 mg / L, 470 mg / L, 480 mg / L, 490 mg / L, 500 mg / L and any range composed of any two of these values. To improve the flocculation efficiency of the bait microalgae, preferably, the final concentration of diatomite in the mixed system is 120 - 450 mg / L, more preferably 150 - 360 mg / L, and further preferably 180 - 350 mg / L.

[0044] In the present invention, in order to improve the flocculation efficiency of bait microalgae, the final concentration of the chitosan in the mixed system is 20 - 80 mg / L, for example, it can be 20 mg / L, 25 mg / L, 30 mg / L, 35 mg / L, 40 mg / L, 45 mg / L, 50 mg / L, 55 mg / L, 60 mg / L, 65 mg / L, 70 mg / L, 75 mg / L, 80 mg / L. Preferably, it is 20 - 60 mg / L, and more preferably, it is 25 - 55 mg / L.

[0045] In the present invention, the weight ratio of the chitosan to the diatomaceous earth is not particularly limited; in order to improve the flocculation efficiency of bait microalgae, preferably, the weight ratio of the chitosan to the diatomaceous earth is 1:(1 - 15), for example, it can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15 and any range composed of any two of these values. Considering the flocculation efficiency and harvesting cost comprehensively, the weight ratio of the chitosan to the diatomaceous earth is 1:(3 - 9).

[0046] In the present invention, in order to improve the flocculation efficiency of bait microalgae, preferably, the final concentration of the diatomaceous earth and the chitosan in the mixed system is 120 - 520 mg / L, for example, it can be 120 mg / L, 130 mg / L, 140 mg / L, 150 mg / L, 160 mg / L, 170 mg / L, 180 mg / L, 190 mg / L, 200 mg / L, 210 mg / L, 220 mg / L, 230 mg / L, 240 mg / L, 250 mg / L, 260 mg / L, 270 mg / L, 280 mg / L, 290 mg / L, 300 mg / L, 310 mg / L, 320 mg / L, 330 mg / L, 340 mg / L, 350 mg / L, 360 mg / L, 370 mg / L, 380 mg / L, 390 mg / L, 400 mg / L, 410 mg / L, 420 mg / L, 430 mg / L, 440 mg / L, 450 mg / L, 460 mg / L, 470 mg / L, 480 mg / L, 490 mg / L, 500 mg / L, 510 mg / L, 520 mg / L and any range composed of any two of these values. In order to make the flocculation efficiency higher and reduce the harvesting cost, preferably, the final concentration of the diatomaceous earth and the chitosan in the mixed system is 180 - 450 mg / L, and more preferably, it is 260 - 400 mg / L.

[0047] In the present invention, in order to improve the flocculation efficiency of bait microalgae, preferably, the chitosan is acid-soluble chitosan.

[0048] In the present invention, the diatomite and the chitosan are commercially available conventional diatomite and chitosan.

[0049] In the present invention, the diatomite and the chitosan can be first mixed to form a mixture and then added to the microalgae culture solution. The mixing method can be to mix the acid-soluble chitosan and the diatomite in water containing acetic acid, where the addition amount of the acetic acid can be such that the acid-soluble chitosan can be dissolved. For example, 1 - 20 ml of acetic acid and 1 - 20 g of acid-soluble chitosan can be added to 100 - 1000 mL of water, and then the diatomite is added; the dosage of the diatomite is not particularly limited, and it is sufficient that the final concentration of the mixture of the diatomite and the chitosan in the mixing system is 120 - 520 mg / L. The mixing can be carried out using a shaker or a stirrer, and the mixing time is not particularly limited and can be 10 - 90 min (such as 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min and any range composed of any two of these values), and the rotation speed of the shaker is not particularly limited and can be 50 - 200 rpm (such as 50 rpm, 60 rpm, 80 rpm, 100 rpm, 120 rpm, 140 rpm, 160 rpm, 180 rpm, 200 rpm and any range composed of any two of these values).

[0050] In the present invention, there is no particular limitation on the type of the ferric ion compound, and it can be various ferric ion compounds commonly used in the art. In order to further improve the harvesting effect of the bait microalgae, preferably, the ferric ion compound is selected from at least one of ferric chloride, ammonium ferric citrate, iron EDTA, ferric nitrate and ferric sulfate.

[0051] In the present invention, in order to further improve the harvesting effect of the bait microalgae and minimize the introduction of metal ions, preferably, the final concentration of the ferric ion in the mixing system is 220 - 350 mg / L, for example, it can be 220 mg / L, 230 mg / L, 240 mg / L, 250 mg / L, 260 mg / L, 270 mg / L, 280 mg / L, 290 mg / L, 300 mg / L, 310 mg / L, 320 mg / L, 330 mg / L, 340 mg / L, 350 mg / L and any range composed of any two of these values, and more preferably 220 - 300 mg / L.

[0052] According to a preferred embodiment of the present invention, the method comprises: (1) mixing diatomite and chitosan with the culture solution of bait microalgae; (2) introducing a ferric ion compound into the mixture for sedimentation. The sedimentation time is 10 - 30 min; the sedimentation temperature is 15 - 50 °C; in the culture solution of the bait microalgae, the culture period of the bait microalgae is any culture period; the initial OD value of the culture solution of the bait microalgae is 0.5 - 3; the bait microalgae is at least one of Tetraselmis sp., Phaeodactylum tricornutum, Nitzschia sp., Chaetoceros sp., Skeletonema costatum, Isochrysis galbana, Heterogloea sp., Nannochloropsis sp., Chlorella sp.; the final concentration of diatomite in the mixed system is 120 - 450 mg / L; the final concentration of chitosan in the mixed system is 20 - 80 mg / L; the final concentration of diatomite and chitosan in the mixed system is 120 - 520 mg / L; the ferric ion compound is selected from at least one of ferric chloride, ammonium ferric citrate, iron EDTA, ferric nitrate and ferric sulfate; the final concentration of ferric ions in the mixed system is 220 - 350 mg / L.

[0053] According to a particularly preferred embodiment of the present invention, the method comprises: (1) mixing diatomite and chitosan with the culture solution of bait microalgae; (2) introducing a ferric ion compound into the mixture for sedimentation. The sedimentation time is 10 - 30 min; the sedimentation temperature is 15 - 50 °C; in the culture solution of the bait microalgae, the culture period of the bait microalgae is any culture period; the initial OD value of the culture solution of the bait microalgae is 0.5 - 3; the bait microalgae is at least one of Tetraselmis sp., Phaeodactylum tricornutum, Nitzschia sp., Chaetoceros sp., Skeletonema costatum, Isochrysis galbana, Heterogloea sp., Nannochloropsis sp., Chlorella sp.; the final concentration of diatomite in the mixed system is 180 - 350 mg / L; the final concentration of chitosan in the mixed system is 20 - 60 mg / L; the final concentration of diatomite and chitosan in the mixed system is 260 - 400 mg / L; the ferric ion compound is selected from at least one of ferric chloride, ammonium ferric citrate, iron EDTA, ferric nitrate and ferric sulfate; the final concentration of ferric ions in the mixed system is 220 - 350 mg / L.

[0054] The second aspect of the present invention provides the application of the method described in the first aspect of the present invention in aquaculture.

[0055] In the present invention, the bait microalgae obtained by the harvesting method can be used to feed shellfish, shrimp larvae and some fish larvae, and secondary bait organisms of freshwater and seawater zooplankton in aquaculture.

[0056] The present invention will be described in detail below. The raw materials and reagents used in the present invention are all commercially available products.

[0057] Isochrysis galbana was purchased from the Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences, with the strain number FACHB-861;

[0058] Nannochloropsis oceanica was purchased from the Aquaculture Research Center of the Institute of Marine and Environmental Technology, Center for Environmental Science of the University of Maryland, with the strain number IMET1.

[0059] Tetraselmis subcordiformis was purchased from the Algae Culture Collection of Ocean University of China, with the strain number T.subcordiformis.

[0060] Phaeodactylum tricornutum was obtained by screening through a project cooperation between Guotou Biotechnology Investment Co., Ltd. and the Institute of Hydrobiology, Chinese Academy of Sciences, and was preserved in the Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences, with the strain number FACHB-842;

[0061] Chlorella pyrenoidosa was purchased from the Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences, with the strain number FACHB-2.

[0062] Measurement of the optical density (OD value) of the microalgae culture solution: The optical density value was measured using a spectrophotometer. Using fresh medium as a control, the absorbance value of the microalgae culture solution at a wavelength of 680 nm (Isochrysis galbana, Phaeodactylum tricornutum) or 750 nm (Chlorella pyrenoidosa, Tetraselmis subcordiformis, Nannochloropsis oceanica) was measured as an index of the microalgae concentration.

[0063] Flocculation efficiency (%) = (Initial OD value - OD value of the supernatant after sedimentation) / Initial OD value.

[0064] Acid-soluble chitosan was purchased from Sinopharm Chemical Reagent Co., Ltd., with the brand name of Shanghai Reagent.

[0065] Water-soluble chitosan was purchased from Henan Zhigao Biotechnology Co., Ltd.

[0066] Diatomaceous earth was purchased from Tianjin Dengfeng Chemical Reagent Factory.

[0067] Preparation Example 1

[0068] This preparation example is used to illustrate the preparation of the diatomaceous earth and chitosan mixture G1.

[0069] Weigh 1 g of acid-soluble chitosan and put it into 100 mL of pure water. Then, while stirring, add 1 mL of glacial acetic acid. After complete dissolution, add 6 g of diatomaceous earth, place it on a shaker with a rotation speed of 120 rpm, and use it after 30 minutes.

[0070] (1) Influence of initial algal solution concentration on the flocculation efficiency of Isochrysis galbana

[0071] Isochrysis galbana selected from the late logarithmic phase was added with a flocculant while being slowly stirred. First, a mixture of diatomite and chitosan (G1) was added, and after stirring evenly, FeCl 3 was added. After stirring for 5 min, it was allowed to settle for 20 min; the final concentration of the diatomite and chitosan mixture was 350 mg / L, and the final concentration of FeCl 3 was 250 mg / L.

[0072] The initial OD 680 values were set to 0.5, 1, 1.5, 2, and 2.5 respectively. After the settlement ended, the final OD 680 of the algal solution was measured, and the corresponding flocculation efficiency was calculated. The results are shown in Figure 1 .

[0073] When the culture period of Isochrysis galbana, the concentration of the flocculant, and the settlement time were fixed, when the initial OD 680 value of Isochrysis galbana was 0.5 - 2.5, the flocculation efficiency could reach over 75%. When the initial OD 680 value was 1 - 2, the flocculation efficiency was higher; considering the initial algal solution concentration in actual large-scale farming and the commercial application of the harvesting method, Isochrysis galbana with an initial OD 680 value of 1.5 was finally selected for harvesting.

[0074] (2) Influence of the culture period of bait microalgae on the flocculation efficiency of Isochrysis galbana

[0075] The initial OD 680 value of the Isochrysis galbana algal solution was 1.5. A flocculant was added while being slowly stirred. First, a mixture of diatomite and chitosan (G1) was added and stirred for 10 s, then FeCl 3 was added. After stirring for 5 min, it was allowed to settle for 20 min; the final concentration of the diatomite and chitosan mixture was 350 mg / L, and the final concentration of FeCl 3 was 250 mg / L.

[0076] The culture periods of Isochrysis galbana were the lag phase, early logarithmic phase, middle logarithmic phase, late logarithmic phase, or stationary phase respectively. After the settlement ended, the final OD 680 of the algal solution was measured, and the corresponding flocculation efficiency was calculated. The results are shown in Figure 2 .

[0077] As can be seen from Figure 2 , the flocculation efficiencies of Isochrysis galbana in different culture periods were all above 85% and the differences were not significant, indicating that the growth period had little effect on the flocculation effect of the algal solution. Finally, considering that the microalgae harvested in actual production are mostly those in the late logarithmic phase, Isochrysis galbana in the late logarithmic phase was finally selected for subsequent experiments.

[0078] (III) Influence of the final concentration of FeCl 3 on the flocculation efficiency of Isochrysis galbana

[0079] Isochrysis galbana selected from the late logarithmic phase, with the initial OD 680 value of 1.5. While slowly stirring, the flocculant was added. First, the mixture of diatomite and chitosan (G1) was added and stirred for 10 s, and then FeCl 3 was added. After stirring for 5 min, it was allowed to settle for 20 min; the final concentration of the mixture of diatomite and chitosan was 350 mg / L.

[0080] Among them, the final concentrations of FeCl 3 were 125 mg / L, 175 mg / L, 200 mg / L, 225 mg / L, 250 mg / L, and 300 mg / L respectively. After the settlement, the final OD 680 of the algal solution was measured, and the corresponding flocculation efficiency was calculated. The results are shown in Figure 3 .

[0081] It can be seen from Figure 3 the results that the higher the concentration of the trivalent iron ion compound, the better the flocculation efficiency. Especially when the concentration is in the range of 225 - 300 mg / L, the flocculation efficiency is higher than 75%. However, in order to minimize the cost and reduce the introduction of metal ions on the premise of relatively high flocculation efficiency, the final concentration of FeCl 3 was finally selected as 250 mg / L.

[0082] (IV) Influence of the composition of the mixture of diatomite and chitosan on the flocculation efficiency of Isochrysis galbana

[0083] Isochrysis galbana selected from the late logarithmic phase, with the initial OD 680 value of 1.5. While slowly stirring, the flocculant was added. First, the mixture of diatomite and chitosan was added and stirred for 10 s, and then FeCl 3 was added. After stirring for 5 min, it was allowed to settle for 20 min; the final concentration of FeCl 3 was 250 mg / L.

[0084] Keeping the weight of the acid-soluble chitosan unchanged, diatomite was added so that its weight was 3 times, 6 times, and 9 times that of the acid-soluble chitosan respectively. The final concentrations of the mixture of chitosan and diatomite were 200 mg / L, 350 mg / L, and 500 mg / L respectively. After the settlement, the final OD 680 of the algal solution was measured, and the corresponding flocculation efficiency was calculated. The results are shown in Figure 4 .

[0085] It can be seen from Figure 4As can be seen from the results, the ratio of diatomaceous earth to chitosan has little effect on the flocculation effect of the algal solution. In the harvesting method, when the weight ratio of chitosan to diatomaceous earth is 1:(3 - 9), the flocculation efficiency is above 89%. The harvesting methods using the chitosan and diatomaceous earth mixture with the said weight ratio all have better harvesting effects.

[0086] Considering the cost and flocculation efficiency comprehensively, G1 with a weight ratio of chitosan to diatomaceous earth of 1:6 was finally selected for the subsequent experiments.

[0087] (V) Influence of the final concentration of the diatomaceous earth and chitosan mixture on the flocculation efficiency of Isochrysis galbana

[0088] Isochrysis galbana selected from the late logarithmic phase, with an OD 680 value of 1.5. While slowly stirring, the flocculant was added. First, the diatomaceous earth and chitosan mixture (G1) was added and stirred for 10 s, and then FeCl 3 was added. After stirring for 5 min, it was allowed to settle for 20 min; the final concentration of FeCl 3 was 250 mg / L.

[0089] Among them, the final concentrations of the diatomaceous earth and chitosan mixture were 70 mg / L, 140 mg / L, 210 mg / L, 280 mg / L, 350 mg / L, 420 mg / L, and 560 mg / L respectively. After the settlement ended, the final OD of the algal solution was measured 680 , and the corresponding flocculation efficiency was calculated. The results are shown in Figure 5 .

[0090] From Figure 5 the results, it can be seen that when the weight ratio of chitosan to diatomaceous earth is 1:6 and the final concentration of the diatomaceous earth and chitosan mixture is 70 - 350 mg / L, the flocculation efficiency increases with the increase in the concentration of the diatomaceous earth and chitosan mixture. When the concentration of the diatomaceous earth and chitosan mixture continues to increase, the flocculation efficiency begins to decline; when the final concentration of the diatomaceous earth and chitosan mixture is 140 - 420 mg / L, the flocculation efficiency is higher than 75%; considering the cost and flocculation efficiency comprehensively, the final concentration of the diatomaceous earth and chitosan mixture was selected as 350 mg / L.

[0091] (VI) Influence of the addition order and the composition of the flocculant on the flocculation effect

[0092] (1) This experimental group is used to illustrate the influence of only adding FeCl 3 on the flocculation efficiency.

[0093] Isochrysis galbana selected from the late logarithmic phase, with an OD 680 value of 1.5. While slowly stirring, the flocculant was added. Only FeCl 3 was added, and after stirring for 5 min, it was allowed to settle for 20 min; FeCl 3The final concentration is 250 mg / L. Measure the final OD of the algal solution 680 , calculate the corresponding flocculation efficiency, and the results are shown in Figure 6 .

[0094] (2) This experimental group is used to illustrate the effect of adding only acid-soluble chitosan on the flocculation efficiency.

[0095] Isochrysis galbana selected from the late logarithmic phase, with an OD680 value of 1.5. Add the flocculant while slowly stirring, and stir only with acid-soluble chitosan. After stirring for 5 min, settle for 20 min. The final concentration of acid-soluble chitosan is 700 mg / L. Measure the final OD of the algal solution 680 , calculate the corresponding flocculation efficiency, and the results are shown in Figure 6 .

[0096] (3) This experimental group is used to illustrate the effect of adding only a mixture of acid-soluble chitosan and diatomaceous earth on the flocculation efficiency.

[0097] (3-1) Isochrysis galbana selected from the late logarithmic phase, with an OD680 value of 1.5. Add the flocculant while slowly stirring, and stir only with a mixture of acid-soluble chitosan and diatomaceous earth (G1). After stirring for 5 min, settle for 20 min. The final concentration of the mixture of acid-soluble chitosan and diatomaceous earth is 700 mg / L. Measure the final OD of the algal solution 680 , calculate the corresponding flocculation efficiency, and the results are shown in Figure 6 .

[0098] (3-2) According to the method in (2-1), the difference is that the final concentration of the mixture of acid-soluble chitosan and diatomaceous earth is 350 mg / L, and the measured flocculation efficiency is 5.4%.

[0099] (4) This experimental group is used to illustrate the effect of adding only chitosan and FeCl 3 on the flocculation efficiency.

[0100] (4-1) Isochrysis galbana selected from the late logarithmic phase, with an OD 680 value of 1.5. Add the flocculant while slowly stirring. First, stir with acid-soluble chitosan for 10 s, and then add FeCl 3 . The final concentration of acid-soluble chitosan is 50 mg / L. After stirring for 5 min, settle for 20 min; the final concentration of FeCl 3 is 250 mg / L. Measure the final OD of the algal solution 680 , calculate the corresponding flocculation efficiency, and the results are shown in Figure 6 .

[0101] (4-2) According to the method in (4-1), the difference is that the final concentration of acid-soluble chitosan is 350 mg / L, and the measured flocculation efficiency is 5.0%.

[0102] (5) This experimental group is used to illustrate the effect of adding only diatomaceous earth and FeCl 3 on the flocculation efficiency.

[0103] Isochrysis galbana selected from the late logarithmic phase, with an OD 680 of 1.5. While slowly stirring, add the flocculant. First add diatomaceous earth and stir for 10 s, then add FeCl 3 , stir for 5 min and then settle for 20 min; the final concentration of diatomaceous earth is 300 mg / L, and the final concentration of FeCl 3 is 250 mg / L. Measure the final OD 680 of the algal solution, calculate the corresponding flocculation efficiency, and the results are shown in Figure 6 .

[0104] (6) This experimental group is used to illustrate the effect of the addition sequence on the flocculation efficiency.

[0105] Isochrysis galbana selected from the late logarithmic phase, with an OD 680 value of 1.5. While slowly stirring, add the flocculant. The addition sequence is to first add FeCl 3 and stir for 10 s, then add the mixture of acid-soluble chitosan and diatomaceous earth (G1), stir for 5 min and then settle for 20 min. The final concentration of G1 is 350 mg / L, and the final concentration of FeCl 3 is 250 mg / L. Measure the final OD 680 of the algal solution, calculate the corresponding flocculation efficiency, and the results are shown in Figure 6 .

[0106] (7) This experimental group is used to illustrate the effect of replacing FeCl 3 with alum on the flocculation efficiency.

[0107] Isochrysis galbana selected from the late logarithmic phase, with an OD 680 value of 1.5. While slowly stirring, add the flocculant. First add the mixture of acid-soluble chitosan and diatomaceous earth (G1) and stir for 10 s, then add alum, stir for 5 min and then settle for 20 min; the final concentration of the mixture of diatomaceous earth and chitosan (G1) is 350 mg / L, and the final concentration of alum is 250 mg / L. Measure the final OD 680 of the algal solution, calculate the corresponding flocculation efficiency, and the results are shown in Figure 6 .

[0108] (8) This experimental group is used to illustrate the effect of replacing acid-soluble chitosan with water-soluble chitosan on the flocculation efficiency.

[0109] Prepare a mixture of diatomaceous earth and chitosan, with a weight ratio of diatomaceous earth to water-soluble chitosan of 6:1, to obtain the chitosan-diatomaceous earth mixture D1.

[0110] Isochrysis galbana selected from the late logarithmic phase, with an OD680 The value is 1.5. While slowly stirring, add the flocculant. First add D1 and stir for 10 s, then add FeCl 3 , stir for 5 min and then settle for 20 min; The final concentration of FeCl 3 is 250 mg / L, and the final concentration of D1 is 350 mg / L. Measure the final OD of the algal solution 680 , calculate the corresponding flocculation efficiency, and the results are shown in Figure 6 .

[0111] (9) This experimental group is used to illustrate the effect of replacing diatomite with sodium-based bentonite on the flocculation efficiency.

[0112] Prepare a mixture of sodium-based bentonite and chitosan. The weight ratio of sodium-based bentonite to acid-soluble chitosan is 6:1 to obtain the chitosan and sodium-based bentonite mixture D2.

[0113] Isochrysis galbana selected from the late logarithmic phase, OD 680 The value is 1.5. While slowly stirring, add the flocculant. First add D2 and stir for 10 s, then add FeCl 3 , stir for 5 min and then settle for 20 min; The final concentration of FeCl 3 is 250 mg / L, and the final concentration of D2 is 350 mg / L. Measure the final OD of the algal solution 680 , calculate the corresponding flocculation efficiency, and the results are shown in Figure 6 .

[0114] (10) This experimental group is used to illustrate the effect of replacing diatomite with calcium-based bentonite on the flocculation efficiency.

[0115] Prepare a mixture of calcium-based bentonite and chitosan. The weight ratio of calcium-based bentonite to acid-soluble chitosan is 6:1 to obtain the chitosan and calcium-based bentonite mixture D3. Measure the final OD of the algal solution 680 , calculate the corresponding flocculation efficiency, and the results are shown in Figure 6 .

[0116] Isochrysis galbana selected from the late logarithmic phase, OD 680 The value is 1.5. While slowly stirring, add the flocculant. First add D1 and stir for 10 s, then add FeCl 3 , stir for 5 min and then settle for 20 min; The final concentration of FeCl 3 is 250 mg / L, and the final concentration of D3 is 350 mg / L.

[0117] Consisted of Figure 6The results show that the composition of the flocculant and the addition sequence of the flocculant have a great influence on the flocculation efficiency of Isochrysis galbana. Under the same other harvesting conditions, when using the flocculant with the above composition for harvesting, the flocculation efficiency is lower than that of the mixture of diatomite and chitosan with a final concentration of 350 mg / L and FeCl 3 composed flocculant;

[0118] When only adding chitosan and FeCl 3 , the flocculation efficiency is lower than 61%, and increasing the final concentration of chitosan increases, but the flocculation efficiency decreases instead, indicating that the harvesting method can improve the flocculation efficiency of microalgae within the scope of the claims.

[0119] Adding FeCl first 3 and then adding the harvesting efficiency of the diatomite and chitosan mixture (Group 6) is much lower than adding the diatomite and chitosan mixture first and then adding FeCl 3 harvesting efficiency.

[0120] (VII) Flocculation effects of different bait microalgae

[0121] After the above experiments, considering that in large-scale production, harvesting usually starts when the algal liquid grows to the late logarithmic phase, the optimal method for harvesting bait microalgae is determined as follows: the culture period of the bait microalgae is the late logarithmic phase; the initial algal liquid concentration OD 680 value is 1.5; add diatomite and chitosan first, and then add FeCl 3 ; the weight ratio of diatomite to chitosan is 6:1; the final concentration of diatomite and chitosan in the mixed system is 350 mg / L; the final concentration of FeCl 3 in the mixed system is 250 mg / L; the sedimentation time is 20 min.

[0122] Using the above optimal method to harvest different types of bait microalgae, the microalgae are selected from Tetraselmis subcordiformis, Phaeodactylum tricornutum, Nannochloropsis oceanica, Isochrysis galbana, Chlorella pyrenoidosa.

[0123] After sedimentation, measure the final OD value of the algal liquid and calculate the corresponding flocculation efficiency. The results are shown in Figure 7 .

[0124] Through Figure 7 the results, it can be seen that using the optimal harvesting method of the present invention to harvest different types of microalgae, the harvesting efficiency is relatively high.

[0125] Moreover, using the method of the present invention, it is not easy to damage the nutritional value of the bait microalgae during the process of harvesting the bait microalgae.

[0126] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including combinations of each technical feature in any other suitable manner. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A method for harvesting bait microalgae, characterized in that, the method comprises: (1) Mixing diatomite and chitosan with the bait microalgae culture solution for flocculation; (2) Introducing a ferric ion compound into the mixed solution for sedimentation.

2. The method according to claim 1, wherein, the mixing time is 3 - 5 min; Preferably, the sedimentation time is 5 - 40 min, preferably 10 - 30 min, more preferably 15 - 25 min; Preferably, the sedimentation temperature is above 0 °C, preferably 15 °C - 50 °C, more preferably 20 - 25 °C.

3. The method according to claim 1 or 2, wherein, the culture period of the bait microalgae is the lag phase, early logarithmic phase, middle logarithmic phase, late logarithmic phase or stationary phase.

4. The method according to any one of claims 1 - 3, wherein, the initial OD value of the culture solution of the bait microalgae is below 10, preferably below 5, more preferably 0.5 - 3.

5. The method according to any one of claims 1 - 4, wherein, the bait microalgae is at least one of Tetraselmis, Phaeodactylum, Nitzschia, Chaetoceros, Skeletonema, Isochrysis, Heterogloea, Nannochloropsis and Chlorella; Preferably, the bait microalgae is at least one of Tetraselmis, Phaeodactylum, Nannochloropsis, Isochrysis and Chlorella.

6. The method according to claim 5, wherein, the Tetraselmis is at least one of Tetraselmis subcordiformis, Tetraselmis pyriformis and Tetraselmis qingdaoensis; Preferably, the Phaeodactylum is Phaeodactylum tricornutum; Preferably, the Nannochloropsis is at least one of Nannochloropsis oceanica, Nannochloropsis oculata, Nannochloropsis limnetica, Nannochloropsis gaditana, Nannochloropsis salina and Nannochloropsis granulata; Preferably, the Isochrysis is at least one of Isochrysis galbana, Dicrateria inornata and Isochrysis zhanjiangensis; Preferably, the Chlorella is at least one of Chlorella pyrenoidosa, Chlorella vulgaris, Chlorella ellipsoidea, Chlorella sorokiniana and Chlorella zofingiensis.

7. The method according to any one of claims 1 - 6, wherein, the final concentration of the diatomite in the mixed system is 100 - 500 mg / L, preferably 120 - 450 mg / L, more preferably 150 - 360 mg / L, further preferably 180 - 350 mg / L; Preferably, the final concentration of the chitosan in the mixed system is 20 - 80 mg / L, preferably 20 - 60 mg / L, more preferably 25 - 55 mg / L; Preferably, the final concentration of the diatomite and chitosan in the mixed system is 120 - 520 mg / L, preferably 180 - 450 mg / L, more preferably 260 - 400 mg / L.

8. The method according to any one of claims 1 - 7, wherein the chitosan is acid - soluble chitosan.

9. The method according to any one of claims 1 - 8, wherein, the ferric ion compound is selected from at least one of ferric chloride, ammonium ferric citrate, iron(III) - EDTA, ferric nitrate and ferric sulfate; Preferably, the final concentration of ferric ions in the mixed system is 220 - 350 mg / L, more preferably 220 - 300 mg / L.

10. Use of the method according to any one of claims 1 - 9 in aquaculture.

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