Anti-flagellate predation of chlorella and screening method thereof
By using a screening method involving co-culturing Chlorella with flagellates and verifying resistance, Chlorella resistant to flagellate predation was obtained, solving the problem of easy contamination in the Chlorella culture system and achieving stable and efficient production results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHENZHEN UNIVERSITY OF ADVANCED TECHNOLOGY
- Filing Date
- 2026-04-09
- Publication Date
- 2026-07-24
AI Technical Summary
Existing technologies are insufficient to effectively enhance Chlorella's resistance to flagellate predation, leading to easy contamination of the culture system, resulting in economic losses and resource waste. Furthermore, existing control methods are energy-intensive, and chemical residues affect product safety.
By co-culturing Chlorella with flagellates, purifying and expanding the culture, and verifying its resistance, stable Chlorella resistant to flagellate predation was screened out. The specific steps included co-culturing, purification in selective culture medium, and resistance verification. The screening was repeated until resistant Chlorella was obtained.
Significant and stable resistance to flagellate predation of Chlorella was obtained, which improved the stability and production efficiency of the culture system, reduced the use of chemical agents, and lowered production costs.
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Figure CN122445472A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant biotechnology, specifically relating to a method for screening Chlorella that is resistant to flagellates preying on it. Background Technology
[0002] Chlorella, as a type of photosynthetic microorganism, is a crucial cellular factory for producing biofuels, high-value nutrients (such as omega-3 fatty acids), feed additives, and for environmental remediation. It also serves as an important biomanufacturing chassis cell. Achieving large-scale, high-density cultivation of Chlorella is a core prerequisite for its industrialization. However, open or semi-open photobioreactor systems are highly susceptible to protozoan contamination, with planktonic protozoa, primarily flagellates, causing the most severe damage. Flagellates feed on Chlorella, reproduce rapidly, and can deplete the exponentially growing population of Chlorella within 24-72 hours, leading to culture collapse and resulting in significant economic losses and resource waste.
[0003] Currently, the main technical means to deal with flagellate pollution are to eliminate or inhibit the polluting organisms from the outside. Although these methods have some effect, they require continuous intervention, consume a lot of energy, leave chemical residues that affect the safety of the products, and cannot fundamentally improve the resistance and stability of Chlorella itself. Summary of the Invention
[0004] This invention provides a method for screening Chlorella that resists flagellate predation. The method involves co-culturing Chlorella with flagellates, purifying and expanding the culture, and verifying the resistance to obtain Chlorella with significant and stable resistance to flagellate predation.
[0005] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows: The first aspect of this invention provides a screening method for resistant flagellates to predation on Chlorella, comprising the following steps: S1. Chlorella and flagellates are inoculated into the same culture medium at a preset ratio and cultured for a certain period of time to allow the flagellates to fully prey on the chlorella. S2. When the number of Chlorella drops to a preset threshold and then begins to rise, take the culture medium containing Chlorella and spread it on a selective culture medium to remove flagellates. S3. Pick a single colony of Chlorella from the selective culture medium and purify it by streak plate method or limiting dilution method to obtain monoclonal Chlorella. S4. Expand the monoclonal Chlorella culture and use the flagellates to verify the resistance of the expanded monoclonal Chlorella. S5. If the expanded monoclonal Chlorella possesses resistance to flagellate predation, then flagellate-resistant Chlorella is obtained; if the expanded monoclonal Chlorella does not possess resistance to flagellate predation, then repeat steps S1 to S4 until the expanded monoclonal Chlorella possesses resistance to flagellate predation, thus obtaining flagellate-resistant Chlorella.
[0006] As can be seen from the above technical solutions, the screening method for Chlorella resistant to flagellate predation provided by the first aspect of the present invention can obtain Chlorella with significant and stable resistance to flagellate predation through co-culturing Chlorella with flagellates, purification and expansion, and resistance verification. Furthermore, this screening method is highly versatile, simple to operate, requires no expensive equipment, and is convenient for laboratory and industrial applications.
[0007] The second aspect of this invention provides a *Chlorella* species resistant to flagellate predation, the taxonomic name of which is *Chlorella sorokinense* (…). Chlorella sorokiniana SZ-1 was deposited at the China Center for Type Culture Collection on February 2, 2026, with accession number CCTCC NO: M2026331; the anti-flagellate Chlorella was obtained by the screening method described in any embodiment of the present invention, and the Chlorella in S1 was selected from Chlorella sorokinense ( Chlorella sorokiniana ).
[0008] As can be seen from the above technical solutions, the Chlorella anti-flagellate provided by the second aspect of the present invention can effectively resist flagellate predation, and the resistance trait is stable. It does not involve gene manipulation and can be directly applied to laboratory and industrial cultivation. It helps to reduce the use of chemical agents, avoid drug residues, improve the stability and production efficiency of the cultivation system, and reduce production costs. It has important application value for the stable and efficient production of Chlorella. Attached Figure Description
[0009] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0010] Figure 1 This is a flowchart illustrating a screening method for resisting flagellate predation of Chlorella provided in some embodiments of the present invention. Figure 2 It is Sorokin Chlorella ( Chlorella sorokiniana GT-1 and Quantity change curves during co-cultivation; Figure 3 It is Sorokin Chlorella (Chlorella sorokiniana Figure showing the alignment results of the 18S rRNA gene sequence of SZ-1; Figure 4 It is Sorokin Chlorella ( Chlorella sorokiniana GT-1 and Sorokin Chlorella ( Chlorella sorokiniana Comparison of cell morphology of SZ-1; Figure 5 It is Sorokin Chlorella ( Chlorella sorokiniana GT-1 and Sorokin Chlorella ( Chlorella sorokiniana SZ-1 in normal and Growth curve under polluted conditions; Figure 6 It is Sorokin Chlorella ( Chlorella sorokiniana GT-1 and Sorokin Chlorella ( Chlorella sorokiniana Comparison of the biochemical composition of SZ-1. Detailed Implementation
[0011] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0012] The flowchart shown in the attached diagram is for illustrative purposes only and does not necessarily include all content and operations / steps, nor does it necessarily have to be performed in the order described. For example, some operations / steps can be broken down, combined, or partially merged, so the actual execution order may change depending on the actual situation.
[0013] It should be understood that the terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.
[0014] It should also be understood that the term “and / or” as used in this specification and the appended claims refers to any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.
[0015] Chlorella, as a type of photosynthetic microorganism, is a crucial cellular factory for producing biofuels, high-value nutrients (such as omega-3 fatty acids), feed additives, and for environmental remediation. It also serves as an important biomanufacturing chassis cell. Achieving large-scale, high-density cultivation of Chlorella is a core prerequisite for its industrialization. However, open or semi-open photobioreactor systems are highly susceptible to protozoan contamination, with planktonic protozoa, primarily flagellates, causing the most severe damage. Flagellates feed on Chlorella, reproduce rapidly, and can deplete the exponentially growing population of Chlorella within 24-72 hours, leading to culture collapse and resulting in significant economic losses and resource waste.
[0016] Currently, pollution control strategies employed in industrial production and scientific research primarily revolve around "external intervention," and can be categorized into three main types: chemical methods, physical methods, and biological methods. Chemical methods mainly involve the use of antibiotics (such as penicillin and streptomycin) or disinfectants (such as sodium hypochlorite). While these methods are simple to operate, they suffer from problems such as drug residues affecting the safety of downstream products, the potential to induce drug resistance, high costs, and environmental pollution. Physical methods, such as membrane filtration, ultraviolet light, or ultrasonic treatment, can effectively kill or remove pollutants, but generally involve large equipment investments, high energy consumption, limited processing capacity, and the potential to cause physical damage or physiological stress to the Chlorella cells themselves. Biological methods attempt to introduce predators at higher trophic levels (such as specific rotifers) to control flagellate populations, but their controllability is poor, operations are complex, and there is a risk of introducing secondary pollution. In summary, all related technologies aim to eliminate or inhibit polluting organisms from the outside. While these methods have some effect, they require continuous intervention, consume high energy, leave chemical residues affecting product safety, and cannot fundamentally improve the resistance and stability of Chlorella itself.
[0017] In view of this, the present invention provides a Chlorella resistant to flagellate predation and a screening method thereof. The method involves co-culturing Chlorella with flagellates, purifying and expanding the culture, and verifying the resistance to obtain Chlorella with significant and stable resistance to flagellate predation.
[0018] The following detailed description of some embodiments of the present invention is provided in conjunction with the accompanying drawings. Unless otherwise specified, the following embodiments and features can be combined with each other.
[0019] Please see Figure 1 This invention provides a screening method for resisting flagellates preying on Chlorella, specifically including steps S1 to S5.
[0020] Step S1: Inoculate Chlorella and flagellates into the same culture medium at a preset ratio and culture for a certain period of time to allow the flagellates to fully prey on Chlorella.
[0021] Among them, Chlorella is a photosynthetic Chlorella that can be artificially cultured on a large scale, and flagellates include , It is a pleotrophic flagellate. Under light conditions, it can survive through photosynthesis, while when food is plentiful, it can obtain nutrients by ingesting organic particles such as bacteria and chlorella.
[0022] In some embodiments, Chlorella is selected from Sorokin Chlorella ( Chlorella sorokiniana Chlorella proteoglycans () Chlorella pyrenoidosa ), common Chlorella ( Chlorella vulgaris ), Micrococcus ellipsoides ( Chlorella ellipsoidea Chlorella vulgaris ( Chlorella emersonii Chlorella kappa ( Chlorella kesslerii One of them. Among them, Chlorella sorokinense ( Chlorella sorokiniana It possesses characteristics of rapid growth, high temperature resistance, and ease of high-density cultivation, making it a commonly used model species in the industrial production of Chlorella; Chlorella proteoglycans (… Chlorella pyrenoidosa It exhibits outstanding environmental resistance, maintaining stable growth even under fluctuating culture conditions, thus ensuring the stability and repeatability of multiple rounds of screening; *Chlorella vulgaris* (… Chlorella vulgaris With strong adaptability and low cultivation cost, it is suitable for large-scale screening scenarios; Chlorella vulgaris ( Chlorella ellipsoidea Excellent settling properties facilitate separation and purification during the screening process, significantly improving screening efficiency; Chlorella vulgaris ( Chlorella emersonii Its outstanding photosynthetic efficiency can quickly provide sufficient prey for flagellates, ensuring stable and controllable predation pressure in the screening system; Chlorella kwangsiensis ( Chlorella kesslerii It has excellent resistance to algal contamination, which can reduce the interference of algal contamination on experimental results during the screening process and improve the reliability of screening results.
[0023] In some implementations, the initial number of Chlorella is 1 × 10⁻⁶. 6 cells / mL ~1×10 8 cells / mL, with an initial number of flagellates of 1×10⁻⁶. 2 cells / mL ~1×10 7 cells / mL.
[0024] The initial quantity of Chlorella was controlled at 1×10⁻⁶. 6 cells / mL ~1×10 8 A cell / mL ratio provides a sufficient and continuous food source for flagellates while ensuring that Chlorella maintains normal physiological activity and growth, thus contributing to the stability of the co-culture system. For example, the initial number of Chlorella can be 1 × 10⁻⁶ cells / mL. 6 cells / mL, 2×10 6 cells / mL, 3×106 cells / mL, 5×10 6 cells / mL, 5.5×10 6 cells / mL, 6×10 6 cells / mL, 8×10 6 cells / mL, 9×10 6 cells / mL, 1×10 7 cells / mL, 5×10 7 cells / mL, 1×10 8 Typical but not restrictive arbitrary values such as cells / mL, or intervals between any two values.
[0025] The initial number of flagellates was controlled at 1×10. 2 cells / mL ~1×10 7 A cell / mL ratio can exert a moderate and stable predation pressure on Chlorella. For example, the initial number of flagellates could be 1 × 10⁻⁶. 2 cells / mL, 5×10 2 cells / mL, 1×10 3 cells / mL, 6×10 3 cells / mL, 1×10 4 cells / mL, 4×10 4 cells / mL, 1×10 5 cells / mL, 5×10 5 cells / mL, 6×10 5 cells / mL, 8×10 5 cells / mL, 1×10 6 cells / mL, 2×10 6 cells / mL, 1×10 7 Typical but not restrictive arbitrary values such as cells / mL, or intervals between any two values.
[0026] In some implementations, the preset quantity ratio in S1 is (10~10000):1.
[0027] Maintaining the ratio of Chlorella to flagellates between 10 and 10,000:1 ensures sufficient food for the flagellates, maintains the stability of the co-culture system, and applies appropriate predation pressure, which is beneficial for the smooth implementation of multiple rounds of screening. For example, the ratio of Chlorella to flagellates can be any typical but not restrictive value, such as 10:1, 20:1, 50:1, 100:1, 200:1, 500:1, 800:1, 1000:1, 1200:1, 1500:1, 2000:1, 3000:1, 5000:1, 8000:1, or 10000:1, or any range between any two values.
[0028] The culture medium is used for the co-culture of Chlorella and flagellates. The type and volume of the culture medium can be flexibly selected according to the actual application and are not specifically limited here. For example, when Chlorella is selected from Chlorella sorokinense ( Chlorella sorokiniana ), flagellates are When using BG-11 liquid culture medium as the culture medium for co-culture system, a 250 mL Erlenmeyer flask with a culture medium volume of 100 mL can be used.
[0029] In some embodiments, the cultivation conditions in S1 include: a cultivation temperature of 15 ℃ to 35 ℃ and a light intensity of 10 μmol m. -2 s -1 ~1000 μmol m -2 s -1 The cultivation period is 7 to 60 days. Under these conditions, the physiological needs of Chlorella photosynthetic growth and flagellate predation and proliferation can be simultaneously met, maintaining the metabolic activity and overall stability of the co-culture system. For example, the cultivation temperature can be any typical but non-limiting value or a range between any two values, such as 15℃, 16℃, 18℃, 20℃, 21℃, 24℃, 25℃, 28℃, 30℃, and 35℃; the light intensity can be 10 μmol / m². -2 s -1 30 μmol m -2 s -1 40 μmol m -2 s -1 60 μmol m -2 s -1 70 μmol m -2 s -1 100 μmol m -2 s -1 200 μmol m -2 s -1 500 μmol m-2 s -1 700 μmol m -2 s -1 800 μmol m -2 s -1 1000 μmol m -2 s -1 The values can be any typical but non-limiting values or intervals between any two values; the culture time can be any typical but non-limiting value or intervals between any two values, such as 7 days, 10 days, 16 days, 20 days, 25 days, 30 days, 40 days, 50 days, 55 days, 60 days, etc. In some more specific embodiments, the culture conditions in S1 include: a culture temperature of 20 ℃~25 ℃ and a light intensity of 50 μmol / m². -2 s -1 ~500 μmol m -2 s -1 The culture time is 15 days to 40 days.
[0030] Step S2: When the number of Chlorella drops to a preset threshold and then begins to rise again, take the culture medium containing Chlorella and spread it on a selective culture medium to remove flagellates.
[0031] The type of selective culture medium can be flexibly selected according to the actual application, and no specific limitation is made here. For example, when Chlorella is selected from Chlorella sorokinense ( Chlorella sorokiniana ), flagellates are At that time, due to It cannot survive in solid culture medium, therefore BG-11 solid culture medium can be used as a selective medium.
[0032] In some implementations, the preset threshold in S2 is 1% to 5% of the initial number of Chlorella. Controlling the preset threshold within this range ensures that a sufficient number of Chlorella survive after effective predation by flagellates to continue subsequent screening. If the preset threshold is less than 1%, the number of surviving Chlorella may be too low, potentially leading to difficulties in subsequent recovery culture and obtaining a sufficient quantity for the next round of screening. If the preset threshold is greater than 5%, the predation pressure may be insufficient, hindering the complete elimination of Chlorella with weaker predation resistance to flagellates and affecting the enrichment efficiency of the target Chlorella. For example, the preset threshold can be any typical but non-limiting value such as 1%, 1.1%, 1.3%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 4.8%, or 5%, or an interval between any two values.
[0033] Step S3: Pick a single colony of Chlorella from the selective culture medium and purify it by streak plate method or limiting dilution method to obtain monoclonal Chlorella.
[0034] Selecting individual colonies of Chlorella growing independently on selective media ensures that the obtained Chlorella originates from a single algal cell, eliminating interference from other algae. Further purification using streak plating or limiting dilution methods yields monoclonal Chlorella with uniform genetic background and stable traits.
[0035] Step S4: Expand the monoclonal Chlorella culture and use flagellates to verify the resistance of the expanded monoclonal Chlorella.
[0036] For example, monoclonal Chlorella was expanded until a sufficient quantity of Chlorella was obtained to meet the experimental requirements for resistance verification. A column-type photobioreactor was used to culture the expanded monoclonal Chlorella and the same flagellates. The experiment included two control groups and two experimental groups. Control group 1 was inoculated with only the initial Chlorella, and control group 2 was inoculated with only the expanded monoclonal Chlorella. Experimental group 1 was inoculated with both the initial Chlorella and flagellates, and experimental group 2 was inoculated with both the expanded monoclonal Chlorella and flagellates. The culture conditions were consistent with those in step S1. During co-culture, air containing 2% CO2 was continuously introduced into the reactor to ensure carbon source supply and maintain a stable culture environment. The number of Chlorella and flagellates was monitored and recorded daily using a hemocytometer.
[0037] Step S5: If the expanded monoclonal Chlorella possesses resistance to flagellate predation, then flagellate-resistant Chlorella is obtained; if the expanded monoclonal Chlorella does not possess resistance to flagellate predation, then repeat steps S1 to S4 until the expanded monoclonal Chlorella possesses resistance to flagellate predation, thus obtaining flagellate-resistant Chlorella.
[0038] It should be noted that if the expanded monoclonal Chlorella does not have resistance to flagellate predation, steps S1-S4 need to be repeated, and the monoclonal Chlorella expanded in the previous step S4 should be used as the Chlorella in the next step S1, until the expanded monoclonal Chlorella has resistance to flagellate predation, thus obtaining flagellate-resistant Chlorella.
[0039] For example, the resistance verification results show that if the number of expanded monoclonal Chlorella in experimental group 2 continues to increase, while the initial number of Chlorella in experimental group 1 continues to decrease and tends to a very low level, it indicates that the expanded monoclonal Chlorella possesses resistance to flagellate predation. If the number of expanded monoclonal Chlorella in experimental group 2 continues to decrease, it indicates that the expanded monoclonal Chlorella does not possess resistance to flagellate predation. In this case, S1~S4 need to be repeated, and the monoclonal Chlorella expanded in the previous round of step S4 should be used as the Chlorella in the next round of step S1, until the expanded monoclonal Chlorella possesses resistance to flagellate predation.
[0040] In some embodiments, the method further includes: strengthening the resistance trait against flagellates preying on Chlorella, wherein the strengthening steps include: using the Chlorella resistant to flagellate preying obtained in the previous step S4 as the Chlorella in the next step S1, and repeating steps S1 to S4 at least three times.
[0041] By strengthening the resistance trait against flagellates preying on Chlorella, the resistance trait can be made more stable and the resistance level higher.
[0042] As can be seen from the above technical solution, the screening method for Chlorella resistant to flagellate predation of the present invention, through co-culturing Chlorella with flagellates, purification and expansion, and resistance verification, can obtain Chlorella with significant and stable resistance to flagellate predation. Furthermore, this screening method is highly versatile, simple to operate, requires no expensive equipment, and is convenient for laboratory and industrial applications.
[0043] This invention also provides a *Chlorella* species resistant to flagellate predation, the taxonomic name of which is *Chlorella sorokinense* (…). Chlorella sorokiniana SZ-1 was deposited at the China Center for Type Culture Collection on February 2, 2026, with accession number CCTCC NO: M2026331; the anti-flagellate Chlorella was obtained by the screening method as described in any embodiment of the present invention, and the Chlorella in S1 was selected from Chlorella sorokinense ( Chlorella sorokiniana ).
[0044] Sorokin Chlorella ( Chlorella sorokiniana The 18S rRNA gene sequence of SZ-1 is shown in SEQ ID NO.1.
[0045] SEQ ID NO.1:
[0046] Sorokin Chlorella ( Chlorella sorokiniana SZ-1 possesses significant and stable resistance to flagellate predation, enabling it to maintain a growth advantage in the presence of flagellates.
[0047] As can be seen from the above technical solution, the Chlorella anti-flagellate of the present invention can effectively resist flagellate predation, and the resistance trait is stable. It does not involve gene manipulation and can be directly applied to industrial cultivation. It helps to reduce the use of chemical agents, avoid drug residues, improve the stability and production efficiency of the cultivation system, and reduce production costs. It has important application value for the stable and efficient production of Chlorella.
[0048] The following detailed description of the anti-flagellate predation method for Chlorella provided in this invention, along with specific embodiments and experimental data, is provided in detail below.
[0049] Example 1 Please see Figure 1 The specific operation steps in this embodiment are as follows: S1. Set the initial quantity to 1×10 7 Sorokin Chlorella cells / mL Chlorella sorokiniana GT-1 with an initial quantity of 5×10 5 cells / mL Inoculate the culture medium at a ratio of 20:1 into BG-11 liquid medium using 250mL Erlenmeyer flasks. The medium volume is 100mL. Incubate statically under the following conditions: temperature 24℃, light intensity 50 μmol / m². -2 s -1 The cultivation time was 22 days, so that Fully prey on Chlorella; S2, when Sorogenesis ( Chlorella sorokiniana When the GT-1 count drops to 1% of the initial count, take 0.2 mL of Chlorella sorokinense (… Chlorella sorokiniana The culture medium of GT-1 was spread onto BG-11 solid medium to remove... ; S3. Pick *Chlorella sorokinense* from BG-11 solid culture medium ( Chlorella sorokiniana GT-1 single algal colonies were purified by streak plating to obtain monoclonal Chlorella sorokinosa. Chlorella sorokiniana GT-1; S4, Monoclonal Sorokin Chlorella ( Chlorella sorokiniana GT-1 was used for expansion culture, employing The expanded monoclonal Chlorella (Solokin Chlorella) Chlorella sorokiniana GT-1 was used for resistance verification; S5, expanded monoclonal Chlorella vulgaris ( Chlorella sorokiniana GT-1 possesses resistance to flagellate predation, resulting in a flagellate-resistant Chlorella, taxonomically named *Chlorella sorokinense*. Chlorella sorokiniana SZ-1, and its resistance to flagellate predation remains stable after more than 20 generations.
[0050] Experimental methods 1.1 Cell Count The hemocytometer method was used to analyze *Chlorella sorokinense* (…). Chlorella sorokiniana GT 1 and Cell counting was performed, specifically by fixing *Chlorella sorokinica* (Solokin Chlorella) with Lugol reagent after sampling. Chlorella sorokiniana GT 1 and The cells were observed under a microscope and their numbers were quantitatively counted using a hemocytometer.
[0051] 1.2 Algal strain identification Sorokin Chlorella ( Chlorella sorokiniana The 18S rRNA gene sequence of SZ-1 is shown in SEQ ID NO.1. This 18S rRNA gene sequence was submitted to the GenBank database and compared with the published effective algal strain sequences.
[0052] 1.3 Resistance Verification A column-shaped photobioreactor (5 cm inner diameter, 700 mL working volume) was used for the cultivation experiment. Two control groups and two treatment groups were set up: control group 1 was inoculated only with *Chlorella sorokinense* (… Chlorella sorokiniana GT 1. Control group 2 was inoculated only with Chlorella sorokinosa ( Chlorella sorokiniana SZ-1, experimental group 1 was inoculated with Chlorella sorokinosa ( Chlorella sorokiniana GT 1 and Experimental group 2 was inoculated with Chlorella sorokinense ( Chlorella sorokiniana SZ-1 and Each group had three independent biological parallels to ensure the reliability of the statistical data.
[0053] Specifically: Sorokin Chlorella ( Chlorella sorokiniana GT 1. Chlorella sorokinense ( Chlorella sorokiniana SZ-1 and Cells were cultured in BG-11 liquid medium until the logarithmic growth phase, then collected by centrifugation (1000 × g, 5 min) and resuspended in fresh BG-11 liquid medium. *Chlorella sorokinense* (… Chlorella sorokiniana GT 1. With Sorokin Chlorella ( Chlorella sorokiniana The initial quantity of SZ-1 is uniformly adjusted to 1×10 7 cells / mL The initial quantity is adjusted to 3×10 5 cells / mL.
[0054] During co-cultivation, the system temperature was maintained at 22°C, and continuous light was provided (light intensity 100 μmol m). -2 s -1 The light-dark cycle was 24:0, and air containing 2% CO2 was continuously introduced into the reactor to ensure carbon source supply and maintain a stable culture environment. During the experiment, the number of cells in *Chlorella sorokinense* was monitored and recorded daily using a hemocytometer. Chlorella sorokiniana GT 1. Chlorella sorokinense ( Chlorella sorokiniana SZ-1 and The number of cells.
[0055] 1.4 Biochemical Analysis Determination of Chlorella sorokinosa ( Chlorella sorokiniana GT 1. With Sorokin Chlorella ( Chlorella sorokiniana The biochemical composition of SZ-1 includes crude protein content (%), crude lipid content (%), and total carbohydrate content (%). The algal samples collected by centrifugation were freeze-dried to produce algal powder for later use.
[0056] The crude protein content was determined using the modified Bradford method (reagent kit P0006 purchased from Beyotime): 10 mg of algal powder was weighed, extracted with 1 M NaOH, and the supernatant was used for color development with Bradford working solution. The absorbance was measured at 595 nm, and the content was calculated based on the bovine serum albumin standard curve.
[0057] The crude lipid content was determined by the dry weight method: 80 mg of algal powder was weighed and extracted multiple times with 10% dimethyl sulfoxide-methanol solution and diethyl ether-n-hexane solution (1:1). The combined organic phases were washed with water, dried under nitrogen, and weighed for calculation.
[0058] The total carbohydrate content was determined by the sulfuric acid-phenol method: 10 mg of algal powder was weighed, pretreated with glacial acetic acid and acetone, and then hydrolyzed with 4 M trifluoroacetic acid; the hydrolysate was reacted with freshly prepared sulfuric acid-phenol colorimetric solution, and the absorbance was measured at 490 nm after color development at 100 °C. The content was calculated according to the glucose standard curve.
[0059] All measurements were performed in parallel.
[0060] Experimental results Figure 2 It is Sorokin Chlorella ( Chlorella sorokiniana GT-1 and A graph showing the quantitative changes during the co-cultivation process. From... Figure 2 It can be seen that *Chlorella sorokinense* ( Chlorella sorokiniana The amount of GT-1 (i.e., Chlorella) added Then, quickly from 1×10 7 The number of cells / mL decreased to 0.2 × 10⁻⁶. 6 cells / mL; after 4 days, *Chlorella sorokinense* ( Chlorella sorokiniana The number of GT-1 cells gradually increased, and after 20 days, the cell number recovered to 2×10⁻⁶. 6 cells / mL. The quantity changes show the opposite trend. The above results indicate that *Chlorella sorokinense* (…) Chlorella sorokiniana The GT-1 population contains a resistant subpopulation that can resist flagellate predation. This subpopulation proliferates and expands after adaptation, thereby driving the growth of Chlorella sorokinense (…). Chlorella sorokiniana The overall population size of GT-1 has increased.
[0061] like Figure 3 It is Sorokin Chlorella ( Chlorella sorokiniana The image shows the alignment results of the 18S rRNA gene sequence of SZ-1. Figure 3 It can be known that the resistant flagellate-preying Chlorella is *Chlorella sorokinense* (…). Chlorella sorokiniana Its taxonomic name is Sorokin Chlorella ( Chlorella sorokiniana )SZ-1.
[0062] Figure 4 It is Sorokin Chlorella ( Chlorella sorokiniana GT-1 and Sorokin Chlorella ( Chlorella sorokiniana A comparison of cell morphology of SZ-1 cells. Among them, Figure 4 A is *Chlorella sorokinense* ( Chlorella sorokiniana GT-1, Figure 4 B is *Chlorella sorokinense* ( Chlorella sorokiniana SZ-1. From Figure 4 It can be seen that *Chlorella sorokinense* (Chlorella sorokiniana SZ-1 and Sorokin Chlorella ( Chlorella sorokiniana GT-1 cells show very little difference in cell morphology, all being spherical or ellipsoidal single-celled algae with a cell size of 3μm~6μm. Each cell contains a large ring-shaped chloroplast that occupies almost the entire cell, which is consistent with the typical morphological characteristics of Chlorella.
[0063] Figure 5 It is Sorokin Chlorella ( Chlorella sorokiniana GT-1 and Sorokin Chlorella ( Chlorella sorokiniana SZ-1 in normal and Growth curves under polluted conditions. From Figure 5 It can be seen that, under the absence of predation pressure, *Chlorella sorokinense* (… Chlorella sorokiniana GT-1 (i.e., Chlorella GT-1) grows faster than Sorokin Chlorella ( Chlorella sorokiniana SZ-1 (i.e., Chlorella SZ-1), after 4 days of cultivation, *Chlorella sorokinense* ( Chlorella sorokiniana The number of GT-1 cells can reach 1.6 × 10⁻⁶. 8 cells / mL, while the same period of Sorokin Chlorella ( Chlorella sorokiniana SZ-1 has a strength of only 0.9 × 10⁻⁶. 8 cells / mL, after 10 days of culture, *Chlorella sorokinense* ( Chlorella sorokiniana SZ-1 can reach 1.3×10 8 cells / mL. Under predation pressure, *Chlorella sorokinense* ( Chlorella sorokiniana GT-1 cell growth immediately stopped, and the number gradually decreased to extremely low levels; while Sorokin Chlorella ( Chlorella sorokiniana The cell number of SZ-1 continued to increase, and after 10 days of culture, the cell number recovered to the normal level under no-predation-stress conditions. These results indicate that *Chlorella sorokinense* (SZ-1)... Chlorella sorokiniana SZ-1 exhibits significant resistance to flagellate predation.
[0064] Figure 6 It is Sorokin Chlorella ( Chlorella sorokiniana GT-1 and Sorokin Chlorella ( Chlorella sorokiniana A comparison chart of the biochemical composition of SZ-1. (From...) Figure 6 It can be seen that, under the absence of predation pressure, *Chlorella sorokinense* (… Chlorella sorokiniana SZ-1 (i.e., SZ-1) is similar in biochemical composition to Chlorella sorokinense ( Chlorella sorokiniana There is no significant difference compared to GT-1 (i.e., GT-1).
[0065] In summary, the Chlorella obtained by the screening method for Chlorella resistance to flagellate predation provided in the embodiments of the present invention can effectively resist flagellate predation and exhibit stable resistance traits.
Claims
1. A screening method for resistance to flagellates preying on Chlorella, characterized in that, Includes the following steps: S1. Chlorella and flagellates are inoculated into the same culture medium at a preset ratio and cultured for a certain period of time to allow the flagellates to fully prey on the chlorella. S2. When the number of Chlorella drops to a preset threshold and then begins to rise, take the culture medium containing Chlorella and spread it on a selective culture medium to remove flagellates. S3. Pick a single colony of Chlorella from the selective culture medium and purify it by streak plate method or limiting dilution method to obtain monoclonal Chlorella. S4. Expand the monoclonal Chlorella culture and use the flagellates to verify the resistance of the expanded monoclonal Chlorella. S5. If the expanded monoclonal Chlorella possesses resistance to flagellate predation, then flagellate-resistant Chlorella is obtained; if the expanded monoclonal Chlorella does not possess resistance to flagellate predation, then repeat steps S1 to S4 until the expanded monoclonal Chlorella possesses resistance to flagellate predation, thus obtaining flagellate-resistant Chlorella.
2. The screening method according to claim 1, characterized in that, The Chlorella was selected from Chlorella sorokin ( Chlorella sorokiniana Chlorella proteoglycans () Chlorella pyrenoidosa ), common Chlorella ( Chlorella vulgaris ), Micrococcus ellipsoides ( Chlorella ellipsoidea Chlorella vulgaris ( Chlorella emersonii Chlorella kappa ( Chlorella kesslerii One of them.
3. The screening method according to claim 1, characterized in that, The initial quantity of Chlorella was 1×10⁻⁶. 6 cells / mL ~1×10 8 The initial number of flagellates was 1 × 10⁻⁶ cells / mL. 2 cells / mL ~1×10 7 cells / mL.
4. The screening method according to claim 1, characterized in that, The preset quantity ratio in S1 is (10~10000):
1.
5. The screening method according to claim 1, characterized in that, The cultivation conditions in S1 included: a cultivation temperature of 15℃~35℃ and a light intensity of 10 μmol / m². -2 s -1 ~1000 μmol m -2 s -1 The culture time is 7 days to 60 days.
6. The screening method according to claim 5, characterized in that, The cultivation temperature was 20 ℃~25 ℃, and the light intensity was 50 μmol m. -2 s -1 ~500 μmol m -2 s -1 The culture time is 15 days to 40 days.
7. The screening method according to claim 1, characterized in that, The preset threshold in S2 is 1% to 5% of the initial number of Chlorella.
8. The screening method according to claim 1, characterized in that, Also includes: The resistance trait against flagellates preying on Chlorella is enhanced by the following steps: using the Chlorella resistant to flagellate predation obtained in the previous round S4 as the Chlorella in the next round S1, and repeating S1 to S4 at least three times.
9. A method for preventing flagellates from preying on Chlorella, characterized in that, The taxonomic name of the anti-flagellate Chlorella is *Chlorella sorokinense* (…). Chlorella sorokiniana SZ-1 was deposited at the China Center for Type Culture Collection on February 2, 2026, with accession number CCTCC NO:M2026331; The anti-flagellate Chlorella is obtained by the screening method as described in any one of claims 1 to 8, and the Chlorella in S1 is selected from Chlorella sorokinense ( Chlorella sorokiniana ).