Filamentous microalgae with rapid ball gathering and settling characteristics as well as screening method and application of filamentous microalgae

Abstract

The invention belongs to the technical field of microalgae biology, and particularly discloses filamentous microalgae with rapid ball gathering and settling characteristics as well as a screening method and application of the filamentous microalgae. The preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) NO. 46950 The invention also discloses a screening method of the algal strain, and the method comprises the following steps: sampling from the surface of water body sediment, applying settling time pressure in a stepped manner, carrying out enrichment culture, and finally obtaining a purified algal strain by combining a single algal filament picking and purifying technology which extends according to the phototropism of filamentous microalgae. The algal strain can spontaneously form dense algal balls in the growth process, sedimentation is completed within a short time (the sedimentation rate reaches 90% within 10 min), and the harvesting cost and energy consumption in the algae culture process are greatly reduced. The algal strain can be used in the fields of sewage treatment, high-value product production, feed, biomass energy and the like, and has a wide market application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of microbial technology and bioengineering, specifically relating to filamentous microalgae with rapid aggregate sedimentation characteristics, their screening methods, and applications. Background Technology

[0002] Microalgae are an important biological resource, widely used in aquatic feed, food additives, bioenergy, and environmental remediation. However, microalgae cells are typically tiny and have a density close to that of water, resulting in high harvesting costs, accounting for approximately 20%-30% of the total cultivation cost. Traditional harvesting methods (such as centrifugation, filtration, and chemical flocculation) suffer from high energy consumption, expensive equipment, and the potential to damage cells or introduce chemical pollution.

[0003] Filamentous microalgae have certain advantages in harvesting due to their cell morphology; however, not all filamentous algae can settle efficiently, and the settling rate is significantly affected when mixed with bioflocculants such as fungal agents. Achieving rapid, cost-free settling through the inherent characteristics of the organisms themselves is an ideal way to overcome the bottleneck in microalgae harvesting. Currently, although there are reports of spontaneous flocculation or aggregation of microalgae, these are mostly random occurrences, and the settling rates and stability are often insufficient. There is a lack of targeted screening programs for the specific and superior trait of "rapid agglomeration and settling," and a lack of stable algal strains obtained from this.

[0004] Therefore, there is an urgent need in this field for a filamentous microalgae strain with inherent rapid aggregate settling ability and stable characteristics, as well as a method for efficiently screening such strains. Summary of the Invention

[0005] To address the shortcomings of existing technologies, such as the difficulty and high cost of microalgae harvesting, this paper provides a novel filamentous microalgae strain with stable and rapid aggregate sedimentation capabilities, and offers a method for targeted screening of such strains, thereby broadening its application in low-cost industrialization.

[0006] This invention first provides a filamentous microalgae MOP417 that is self-selected and exhibits rapid aggregate sedimentation characteristics; the filamentous microalgae MOP417 is named... Leptolyngbya angustata It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46950, and the deposit date is December 25, 2025.

[0007] The filamentous microalgae provided by this invention ( L. angustata Under normal culture conditions (32~60 μmol / m 2 Under conditions of 25°C and 100°C of light, these filamentous microalgae can spontaneously form regular or irregular algal spheres with a size of 0.2-3.0 mm. L. angustataUnder static conditions, the natural sedimentation rate of its biomass reaches over 90% within 10 minutes, indicating its broad market application value.

[0008] This invention also provides a method for screening filamentous microalgae with rapid aggregate sedimentation characteristics, the specific steps of which are as follows: S1. Static sedimentation operation: collect filamentous microalgae from the sediment surface of natural water bodies: take water samples and add them to the sedimentation column, let them stand to remove the upper suspension, then centrifuge and collect the bottom precipitate, and inoculate the bottom precipitate into BG11 medium, and after static culture, obtain the culture medium. S2. Cyclic enrichment: Take the culture medium obtained in S1 and repeat the above static sedimentation operation according to step S1. The difference is that the water sample is replaced with an equal volume of culture medium, and the static time is shortened to 60-70% of the original static time. After collecting the bottom sediment and centrifuging, it is inoculated into BG11 for further culture to obtain a second culture medium. Take the second culture medium again and repeat this process for N generations, where N is a positive integer. After each repetition, the static time is shortened to 50-60% of the original static time. The static time can eventually be shortened to no more than 0.5 h to obtain the final enriched solution. S3. Purification: Dilute the final enrichment solution and spread it on a solid BG11 medium plate. Pick out a single algal filament that extends outward driven by phototropism of filamentous microalgae and transfer it to BG11 liquid medium for expansion culture to obtain a pure algal strain.

[0009] Preferably, in step S1, the volume ratio of water sample to BG11 culture medium is 1:1; the standing time is 6-8 h; and the centrifugation conditions are 8,000 rpm and 10 min. The sedimentation column is a conical sedimentation column with a height of 20 cm, an outer diameter of 3 cm, and a conical bottom.

[0010] Preferably, the static incubation conditions in step S1 are: 32 μmol / m 2 Culture for 3-4 weeks under conditions of 25°C light, 25°C temperature, and pH 7.0-8.0.

[0011] Preferably, step S2 is repeated N times, where N is 3-4.

[0012] The present invention also provides an algal ball preparation comprising the aforementioned filamentous microalgae and the algal balls formed after cultivation.

[0013] Finally, this invention provides applications of the filamentous microalgae or algal pellets formed therefrom in wastewater treatment, bioproduct production, and biomass energy, as detailed below: a) Applications in wastewater treatment, particularly in the removal of nitrogen, phosphorus, and heavy metals; b) Applications in the production of microalgae biomass for use in feed, food, or health products; c) Applications in the production of specific metabolites, such as phycocyanin, beta-carotene, and polysaccharides; d) Applications in biomass energy production; e) Applications as bioflocculants or biomaterials. Beneficial effects:

[0014] (1) Excellent strain characteristics: The strain provided by the present invention has spontaneous, rapid and efficient aggregate sedimentation ability, and the sedimentation speed is far greater than that of ordinary filamentous microalgae. It can greatly reduce or even eliminate mechanical or chemical harvesting steps, and greatly save energy and costs.

[0015] (2) Stable traits: This aggregate sedimentation characteristic is an intrinsic genetic trait of the algae strain, which remains stable after multiple generations of cultivation and is suitable for industrial application.

[0016] (3) The screening method is highly efficient: The screening method provided by this invention is simple in principle and easy to operate. By applying "sedimentation time selection pressure", it can selectively and rapidly enrich and separate target algal strains from complex algal communities, and has universal reference value.

[0017] (4) Broad application prospects: Thanks to its easy harvesting characteristics, this algal strain has a significant cost advantage in fields that require large-scale cultivation of microalgae and rapid harvesting and separation. Attached Figure Description

[0018] Figure 1 shows the algal strain of the present invention ( L. angustata Microscopic observation of algal filaments (a); Algal strain of the present invention ( L. narrowed Microscopic observation of the aggregates of the algae (b); the algal strain of the present invention ( L. angustata (c) shows the appearance of the aggregated spheres.

[0019] Figure 2 The results are for algal species identification.

[0020] Figure 3 The sedimentation rate of pure microalgae culture medium during a 20-minute standing period is given.

[0021] Figure 4 shows the changes in microalgae biomass (a) and ammonia nitrogen concentration (b) during the wastewater purification process; and the sedimentation rate of microalgae in the wastewater after standing for 2 minutes (c). Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of the invention. Various modifications and variations to the specific embodiments described in this specification are apparent to those skilled in the art without departing from the scope or spirit of the invention. Other embodiments derived from this specification will be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary. Example 1:

[0024] 1. Sample source: Pond on the campus of Jiangsu University.

[0025] 2. Enrichment medium: BG11 medium was used.

[0026] 3. Screening process: S1. Static sedimentation procedure: Take 100 mL of water sample and add it to a conical sedimentation column (20 cm high, 3 cm outer diameter, cone bottom, 150 mL volume). Let it stand for 6 h, and use a thin tube to remove the upper suspension. Centrifuge the bottom precipitate (8000 rpm, 10 min) and inoculate it into 100 mL of fresh BG11 medium. Incubate at 25°C and light intensity of 32 μmol / m² / s for 3 weeks to obtain the culture medium.

[0027] S2. After 3 weeks of culture, take 100 mL of the above culture medium and repeat the above static sedimentation operation as in step S1. The difference is that the 100 mL water sample is replaced with an equal volume of culture medium, and the static time is shortened to 4 h. Collect the bottom biomass, centrifuge it under the same conditions, and inoculate it into BG11 medium for further culture to obtain a secondary culture medium. Repeat this process for 3 generations, shortening the static time by half each time, until the static time can be shortened to 0.5 h, to obtain the final enriched solution.

[0028] 4. Purification: The final enrichment solution was diluted and spread onto solid BG11 medium plates. Outwardly extending algal filaments were picked and transferred to liquid medium for further culture to obtain a pure algal strain, which was then identified as... Leptolyngbya angustata Its algal filament morphology and aggregate morphology are as follows Figure 1 As shown, the algal species identification results are as follows: Figure 2 .

[0029] Example 2: Determination of sedimentation performance of the strain 1. Take the pure algal strain obtained in Example 1. L. angustata The algae were cultured in BG11 medium until the logarithmic growth phase, and then the algal solution at the end of the logarithmic growth phase was taken and gently shaken.

[0030] 2. Take a certain volume (50mL) of the well-shaken algal solution and inject it into a 50mL graduated cylinder, then start timing.

[0031] 3. Observe the descent of the algae-water interface during the settling process at 0, 1, 3, 5, 10, and 20 min, and record the volume (mL) of the lower settling microalgae layer.

[0032] 4. Calculate the sedimentation rate: Sedimentation rate (%) = (1 - Volume of sedimented microalgae layer / Volume of original mixed liquid) × 100%.

[0033] 5. Results: The algal strain of this invention L. angustata Within 10 minutes, the sedimentation rate reached 90%, while the control group, the common filamentous microalgae strain *Hygrophytes fulvidae*, showed a sedimentation rate of 90%. Tribonema minor and Polysylcolepidium Microcoleus vaginatus (Both were obtained from the Freshwater Algae Seed Bank of the Chinese Academy of Sciences and are publicly known materials) Only 13% and 20% (Figure 3).

[0034] Example 3: Application Experiment of Strains in Wastewater Treatment The algal strains screened using this invention ( L. angustata The treatment of municipal domestic sewage (taken from the inlet of the municipal sewage treatment plant in Jingkou District, Zhenjiang City; wastewater COD: 235 mg / L; ammonia nitrogen: 50 mg / L; total phosphorus: 8 mg / L) was carried out simultaneously with *Phyllostachys edulis* (…). T. less ) and Polysylvatica ( M. vaginatus () serves as a control; The procedure was as follows: at 32 μmol / m for 24 h. 2 Microalgae were cultured for 10-14 days under conditions of 25°C light intensity, filtered sterile air flow at a rate of 2.5 L / min for mixing and agitation to reach the logarithmic growth phase, and then collected. L. angustata The algal culture solution, after being washed with deionized water and centrifuged, was inoculated into the wastewater to achieve an initial microalgae concentration of 0.2 g / L dry weight. Further inoculation was carried out at 60 μmol / m³ for 24 h. 2 Under conditions of 25°C light intensity and 2.5 L / min filtered sterile air flow for mixing and stirring, microalgae were used to treat wastewater; microalgae growth and ammonia nitrogen removal efficiency were monitored, and the microalgae sedimentation and separation rate was measured.

[0035] The results are as follows Figure 4 As shown, compared to the control group, the algal strain of this invention exhibited a continuous growth trend in wastewater, with biomass reaching 0.8 g / L dry weight on the seventh day, which is higher than that of *Hygrophytes glomeratus* (…). T. minor ) and Polysylvatica ( M. vaginatus ) 2.7 and 3.2 times the biomass ( Figure 4 a).

[0036] Meanwhile, the algal strain of this invention ( L. angustata It can quickly remove ammonia nitrogen, with an ammonia nitrogen removal rate reaching 98% on day 5, which is higher than that of *Phyllostachys edulis* (…). T. minor ) and Polysectia ( M. vaginatus ) are 46% and 66% higher.

[0037] Moreover, after settling for 2 minutes, the algal strain of this invention ( L. angustata The sedimentation rate reached 62%, while the control group (Xanthophyllus) T. minor ) and Polysylvatica ( M. vaginatus Both were only 2%.

[0038] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still make modifications or equivalent substitutions to the present invention based on their understanding. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A filamentous microalgae with rapid aggregate settling characteristics, characterized in that, The filamentous microalgae are Leptolyngbya angustata The algal strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 46950.

2. The filamentous microalgae according to claim 1, characterized in that, After cultivation, the filamentous microalgae form regular or irregular algal spheres with a size of 0.2-3.0 mm.

3. The filamentous microalgae according to claim 1 or 2, characterized in that, Under static conditions, the biomass of the filamentous microalgae achieves a natural sedimentation rate of over 90% within 10 minutes.

4. A method for screening filamentous microalgae with rapid aggregate settling characteristics as described in any one of claims 1-3, characterized in that, Includes the following steps: S1. Static sedimentation operation: collect filamentous microalgae from the sediment surface of natural water bodies: take water samples and add them to the sedimentation column, let them stand to remove the upper suspension, then centrifuge to collect the bottom precipitate, and inoculate the bottom precipitate into BG11 medium. After static culture, the culture medium is obtained. S2. Cyclic enrichment: Take the culture medium obtained in S1 and repeat the above static sedimentation operation according to step S1. The difference is that the water sample is replaced with an equal volume of culture medium, and the static time is shortened to 60-70% of the original static time. After collecting the bottom sediment and centrifuging, it is inoculated into BG11 for further culture to obtain a second culture medium. Take the second culture medium again and repeat this process for N generations, where N is a positive integer. After each repetition, the static time is shortened to 50-60% of the original static time. The static time can eventually be shortened to no more than 0.5 h to obtain the final enriched solution. S3. Purification: Dilute the final enrichment solution and spread it on a solid BG11 medium plate. Pick out a single algal filament that extends outward driven by phototropism of filamentous microalgae and transfer it to BG11 liquid medium for expansion culture to obtain a pure algal strain.

5. The method according to claim 4, characterized in that, In step S1, the volume ratio of water sample to BG11 culture medium is 1:1; the standing time is 6-8 h; the centrifugation conditions are 8,000 rpm for 10 min; the sedimentation column is a conical sedimentation column with a height of 20 cm, an outer diameter of 3 cm, and a cone-shaped bottom.

6. The method according to claim 4, characterized in that, The static incubation conditions in step S1 are: 32 μmol / m 2 Culture for 3-4 weeks under conditions of 25°C light, 25°C temperature, and pH 7.0-8.

0.

7. The method according to claim 4, characterized in that, In step S2, repeat N generations, where N can be 3-4.

8. An algal ball preparation, characterized in that, It includes the filamentous microalgae and the algal spheres formed therefrom as described in any one of claims 1-3.

9. The use of the filamentous microalgae according to any one of claims 1-3 or the algal ball preparation according to claim 8 in any of the following aspects: a) Applications in wastewater treatment, particularly in the removal of nitrogen, phosphorus, and heavy metals; b) Applications in the production of microalgae biomass for use in feed, food, or health products; c) Applications in the production of specific metabolites, such as phycocyanin, beta-carotene, and polysaccharides; d) Applications in biomass energy production; e) Applications as bioflocculants or biomaterials.

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