Method for cultivating and harvesting of stictophyllum and application thereof in brackish water desalination

Abstract

The application discloses a cultivation and harvesting method of a string algae and application of the string algae in brackish water desalination, and belongs to the technical field of water treatment. Nodosilinea The string algae (Sp.) MH-2 is derived from the intertidal zone of the South China Sea, and has a preservation number of CCTCC NO: M 2026092. The string algae MH-2 is put into brackish water for cultivation, so that the salinity of the brackish water can be effectively reduced. The string algae grows rapidly and has strong salinity and temperature adaptability, and the biological membrane form of the string algae can well solve the industrial bottleneck of difficult microalgae harvesting, and has unique advantages in the field of water treatment. The method has the advantages of simple operation, low cost and environmental friendliness, can efficiently and stably reduce the salinity of brackish water with different salinity, and can easily realize algae-water separation after treatment. The harvested string algae also has high-value utilization potential, and provides a brand-new green solution for resource utilization of brackish water.

Description

Technical Field

[0001] This invention relates to the field of water treatment technology, specifically to a type of nematode (… Nodosilinea sp.) MH-2 and its application in brackish water biological desalination. Background Technology

[0002] Against the backdrop of increasing global water scarcity, brackish water has emerged as a potential alternative water source. However, the direct use of brackish water has serious drawbacks: long-term consumption can lead to health problems such as diarrhea, high blood pressure, and even cardiovascular diseases; its use in agricultural irrigation can cause soil salinization, harming crop growth. Therefore, desalination of brackish water has become one of the key means to solve the problem of freshwater scarcity.

[0003] Existing brackish water desalination technologies, such as reverse osmosis and electrodialysis membrane methods, while highly efficient, suffer from high equipment investment, high energy consumption, and expensive operating costs, making large-scale application difficult in economically underdeveloped regions. Distillation methods are energy-intensive and rarely used anymore. While refrigeration is more energy-efficient than distillation, its treatment effect is easily affected by complex pollutants in the water. Electroadsorption methods have lower energy consumption and are easier to manage, but require high initial investment. Therefore, developing a low-cost, green, and sustainable brackish water desalination technology is urgently needed. In summary, existing desalination technologies are energy-intensive, costly, and environmentally problematic, failing to meet the growing global water demand, necessitating the exploration of more sustainable solutions.

[0004] Biological desalination is a green technology that utilizes salt-tolerant organisms (such as microalgae and microorganisms) to reduce the concentration of salt ions in brackish water through their own absorption and adsorption of salt. Microalgae, as a widely distributed group of photosynthetic autotrophs, can effectively remove nutrients such as nitrogen and phosphorus, as well as heavy metals and organic pollutants from wastewater. They can also regulate the salinity of brackish water through two pathways: first, through active ion absorption via biological regulation systems, such as by regulating the intracellular K⁺ / Na⁺ balance and the Cl⁻ pump mechanism to actively transport inorganic salt ions such as Na⁺ and Cl⁻; second, through adsorption using extracellular polymeric substances (EPS) secreted by algal cells. EPS is mostly negatively charged and contains functional groups such as -COOH and -COO⁻, which can efficiently adsorb cations (such as Na⁺) in water. Studies have confirmed that some microalgae, such as Scenedesmus obliquus (…), can effectively adsorb cations (such as Na⁺). Scenedesmus obliquus The removal capacity of Na⁺ by ) can reach 3.15 g / L, while Chlorella ( Chlorella vulgaris The desalination rate of synthetic brine can reach 40%. These characteristics give microalgae great potential for biological desalination of brackish water.

[0005] However, current research on microalgal desalination mostly employs suspension culture, which has several key bottlenecks: First, microalgal cells are extremely small, resulting in high energy consumption and costs during harvesting, severely limiting its practical application; second, common algal species exhibit poor growth activity and desalination stability under high salt stress. For example, Sahle-Demessie et al. used... Scenedesmus sp. and Chlorella common Two types of freshwater microalgae achieved a salt removal rate of approximately 30% at a salinity of 4 g / L, but at 20 g / L, salinity reduction only occurred after 50 days of cultivation, with a maximum salt removal rate of only 3%. This may be due to the limited salt tolerance of the freshwater algae; under high salt stress, the metabolic activity of algal cells decreases, leading to a sharp decline in salt removal efficiency. Meanwhile, Sahle-Demessie et al. employed a uniform suspension culture mode in their biological desalination process, dispersing algal cells in the water. Harvesting required flocculants for flocculation followed by centrifugation. The introduction of flocculants not only increased reagent costs but also potentially caused secondary pollution to the desalinated water, necessitating additional treatment steps to remove residual flocculants, thus increasing process complexity and operating costs. In addition, the suspension culture mode has high requirements for culture facilities, requiring equipment such as column photobioreactors, circulating pumps, and stirring devices to achieve uniform dispersion of algal solution and utilization of light. It has high energy consumption and is difficult to implement in open water or simple facilities, which limits its large-scale promotion and application (Sahle-Demessie et al., Desalination, 2019, 465: 1-10).

[0006] In summary, although existing research has demonstrated the feasibility of microalgae desalination, overcoming harvesting challenges and obtaining stable and efficient algal strains and processes for treating brackish water remain key obstacles to the engineering application of this technology. Therefore, there is an urgent need in this field for a novel microalgae desalination method that overcomes the aforementioned shortcomings and is efficient, stable, low-cost, and easy to harvest. Summary of the Invention

[0007] In view of the above-mentioned technical defects, the present invention provides a method for utilizing *Nematocystis* (… Nodosilinea A novel method for desalination of brackish water using *Hymenochrysis fusiforme* sp. MH-2. This *Hymenochrysis fusiforme*, isolated from the intertidal zone of the South China Sea, is being used for the first time to treat brackish water with varying salinity. Its unique advantages include: originating from the South China Sea intertidal zone where regional temperature and salinity fluctuate dramatically, it exhibits broad adaptability to temperature and salinity, overcoming the barrier of poor growth stability under varying salinity stress; the algal filaments easily intertwine to form a biofilm, allowing for harvesting through simple and inexpensive filtration methods, effectively solving the problems of high energy consumption and high cost in conventional microalgae harvesting; and its large specific surface area and rapid growth rate ensure the high efficiency of biological desalination of brackish water.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A type of nematode ( Nodosilinea sp.) MH-2, deposited at China Center for Type Culture Collection, accession number: CCTCC NO: M 2026092.

[0010] The aforementioned Nematocystis MH-2 can be used for brackish water desalination.

[0011] A method for desalinating brackish water using Nematocystis aeruginosa includes the following steps:

[0012] (1) Pre-culture: Inoculate Nodidiella MH-2 into freshwater or artificial seawater for pre-culture;

[0013] (2) Collection and washing: After the pre-culture is completed, collect the Nematocystis and wash the Nematocystis with brackish water to be treated;

[0014] (3) Inoculation: Inoculate the washed Nematocystis into the brackish water to be treated;

[0015] (4) Desalination treatment: Cultivate under light conditions and use Nematoda to reduce the salinity of brackish water.

[0016] Furthermore, the method of using Nematocystis aeruginosa to desalinate brackish water specifically includes the following steps:

[0017] (1) Pre-culture of algae: Inoculate Nematocystis MH-2 into the culture medium, such as F / 2 medium, Zarrouk medium, BG11 medium or modified medium SYSU-Lvsan-1 medium, and culture for 1-30 days under the conditions of salinity 0-160‰, temperature 20-40℃ and light intensity 1000-5000 Lux. During the culture process, air or carbon dioxide can be added.

[0018] (2) Collection of Nematocystis: scrape off Nematocystis attached to various materials (including but not limited to glass, plastic, nylon, acrylic, etc.) and use a 300-mesh filter to collect the Nematocystis free in the algal solution and combine the two.

[0019] (3) Washing and inoculation: The collected Nematocystis were filtered and washed 2-3 times with the high-salt brackish water to be treated to remove residual culture medium. The washed Nematocystis were then inoculated into the high-salt brackish water to be treated in a certain proportion.

[0020] (4) Desalination treatment: Cultivate at a temperature of 20-40℃ and a light intensity of 1000-5000 Lux. During the cultivation process, air or carbon dioxide can be added. Take samples daily to monitor the changes in salinity of the brackish water. The treatment continues until the salinity drops to the target value or the change tends to be stable.

[0021] Furthermore, the formulation of the SYSU-Lvsan-1 culture medium is: urea 53.00 mg / L, NaH2PO4·H2O 5.65 mg / L.

[0022] Further, the inoculation ratio described in step (3) is 1-100 g wet Nematocystis thunbergii / L brackish water.

[0023] Furthermore, the cultivation device for the *Nematocystis* includes an open culture container or a closed photobioreactor; the open culture container includes a conical flask, a basin, or a tank; the closed photobioreactor includes an intermittent photobioreactor or a continuous flow photobioreactor.

[0024] Furthermore, the culture device also includes a culture container with a support material selected from glass, plastic, nylon, or acrylic.

[0025] The algae MH-2 was isolated from the intertidal reefs near the South China Sea. Its algal filaments have the characteristic of intertwining to form biofilms, making it easy to harvest. It has a large specific surface area, a fast growth rate, and a strong adaptability to drastic changes in temperature and salinity.

[0026] Because the filaments of *Nematocystis* easily entangle to form biofilms, they readily attach to supports made of various materials (including but not limited to glass, plastic, nylon, acrylic, etc.). Several supports can be placed in the container where *Nematocystis* is cultured, allowing the algae to grow on them. Harvesting is simple: the algae attached to the supports can be collected using a scraper. For algae free in the culture medium, filtration can be used to separate the algae from the water.

[0027] Furthermore, during the desalination process, Nematocystis can effectively reduce the salinity of brackish water through mechanisms such as biosorption, ion exchange, and intracellular accumulation.

[0028] Furthermore, once the salinity of the brackish water is observed to level off, the *Nematocystis* can be removed from the brackish water and replaced with new *Nematocystis* for desalination treatment, thereby further reducing the salinity. Alternatively, some *Nematocystis* can be removed directly to reduce the biomass of *Nematocystis* per unit volume of brackish water, creating conditions for *Nematocystis* to resume growth and further reducing the salinity.

[0029] Furthermore, the Noctuida albicans that has been desalinated once can be inoculated into new, undesalinated brackish water, and the Noctuida albicans can still maintain normal growth, meaning that the Noctuida albicans can be reused.

[0030] Furthermore, the brackish water treated with desalination still contains abundant bioactive substances in the Algae filamentosa. Through simple extraction, bioactive substances such as microalgal polysaccharides, phycocyanin, and polyunsaturated fatty acids can be obtained, which can be used in many fields such as bio-fertilizers, feed, cosmetics, and health product raw materials.

[0031] The beneficial effects of this invention are as follows:

[0032] (1) Solved the harvesting problem: By utilizing the characteristics of Nocturia spontaneously forming biofilm and easily attaching to various materials (including but not limited to glass, plastic, nylon, acrylic, etc.), there is no need for complicated harvesting processes such as centrifugation and flocculation. It can be harvested and reused simply by scraping and filtering, which greatly reduces the cost and energy consumption of subsequent processing.

[0033] (2) High and stable desalination efficiency: The selected algae strains are from the intertidal zone with harsh natural environment. They have strong tolerance and adaptability to changes in salinity and temperature. In the previous experiments, it was found that their salt tolerance range can reach 0-160 g / L. Therefore, they can maintain vigorous growth and high efficiency in desalination in brackish water of various concentrations. They are suitable for desalination of brackish water under different environmental conditions.

[0034] (3) Simple operation and low cost: The method has a simple process, no need to add culture medium or expensive equipment, low requirements for culture facilities, and is suitable for large-scale application. It is a truly green and low-cost biological desalination technology.

[0035] (4) Environmentally friendly: No chemical agents are added during desalination and harvesting, and there is no secondary pollution; the harvested nematodes are rich in a variety of metabolites and can be further utilized as resources, such as extracting bioactive substances, preparing feed or biofuel precursors, to achieve a win-win situation for both the environment and the economy.

[0036] Nematocystis ( Nodosilinea sp.) MH-2 was deposited on January 12, 2026 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, 430072, China, with accession number CCTCC NO: M2026092. Attached Figure Description

[0037] Figure 1 Nematocystis Nodosilinea Morphological characteristics of sp. MH-2.

[0038] Figure 2 Nematocystis Nodosilinea Homology alignment analysis of 16S rDNA sequence of sp. MH-2.

[0039] Figure 3 Nematocystis NodosilineaPhylogenetic tree of the 16S rDNA gene of sp. MH-2. Detailed Implementation

[0040] The following embodiments are further illustrations of the present invention, but not limitations thereof. Specific experimental conditions and methods are not specified in the following embodiments, and the techniques used are generally conventional methods well known to those skilled in the art.

[0041] Example 1

[0042] 1. Isolation of Nematocystis MH-2

[0043] On September 13, 2022, 10 L of seawater was collected from the beach near Tangjiawan, Xiangzhou District, Zhuhai City, Guangdong Province (N 22°20'37'', E 113°35'26''). After initial filtration through a 300-mesh filter to remove sediment, the seawater was then filtered through a 0.22 μm filter membrane to enrich microalgae. The enriched microalgae on the filter membrane were scraped off and transferred to F / 2 medium for cultivation for approximately 20 days. Observation under an optical microscope showed... Figure 1 As shown, the thallus of *Nematocystis MH-2* is filamentous, unbranched, and sometimes forms nodules. It typically possesses a thin, colorless sheath, which is thin, soft, and colorless. The filaments are non-motile and constricted at the transverse walls. The cells are cylindrical, bluish-green, lack air sacs, and possess periphylloids, longer than wide (3.2-5.0 μm long, 1.5-1.8 μm wide). The terminal cells have rounded apexes. There are no heterocysts or thick-walled spores. Reproduction occurs through the fragmentation of the filaments to form thylakoid segments.

[0044] 2. Identification of Nematocystis MH-2

[0045] The algal strain was entrusted to the Freshwater Algae Bank of the Chinese Academy of Sciences for morphological and molecular biological identification. The 16S rDNA fragment sequence of the obtained sample was compared with the GenBank database using the BLAST function. Sequences of similar algae were downloaded from GenBank, and gene sequences were aligned using Clustal X, supplemented by manual correction using SEAVIEW. A phylogenetic tree was constructed using MEGA5 to analyze the phylogenetic position of the sample.

[0046] Sequencing revealed that the 16S rDNA fragment of the sample to be identified was 779 bp in size. Sequence homology comparison between this sequence and data from the NCBi Genbank database showed that this algal strain is related to the genus *Nematocystis* (…). Nodosilinea They are closely related, with sequence homology reaching 97%. Figure 2 ).

[0047] A phylogenetic tree constructed based on the 16S rDNA sequence of the cell ribosomal cells shows that this algal strain Contig 1 is related to the genus Nematocystis (…). NodosilineaThey are closely related and belong to the same evolutionary branch. Figure 3 ).

[0048] Based on a comprehensive analysis of microscopic observation and 16S rDNA sequence sequencing results, the sample was identified as a type of *Nematocystis*. Nodosilinea sp., belonging to the phylum Cyanophyta, class Cyanophyceae, order Nodosilineales, family Nodosilineaceae, genus Nodosiline. Nodosilinea ).

[0049] The isolated and identified algal strain was named *Nematocystis*. Nodosilinea The algal strain MH-2 was deposited on January 12, 2026 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, 430072, with accession number CCTCC NO: M 2026092.

[0050] Example 2

[0051] *Nematocystis* was pre-cultured for 14 days in artificial seawater with a salinity of 20‰. The culture container was a 3 L acrylic tubular photobioreactor, the culture medium was Zarrouk, and the culture conditions were 25℃ and 3000 Lux light intensity. After the pre-culture, the *Nematocystis* adhering to the reactor wall was scraped off, and the remaining *Nematocystis* in the culture medium was collected by filtering through a 300-mesh filter and combined. 10 g of wet weight *Nematocystis* was taken and washed three times with brackish water (salinity 20‰). The washed *Nematocystis* was inoculated into 1 L of brackish water (salinity 20‰) and cultured in a 2 L plastic tank at 25℃ and 3000 Lux light intensity. After 14 days of culture, the salinity of the brackish water decreased from the initial 20‰ to 18‰.

[0052] Example 3

[0053] *Nematocystis* was pre-cultured in freshwater for 7 days in a 5 L acrylic column photobioreactor with four plastic plates inserted inside. The culture medium was BG11, and the culture conditions were 20°C and 1000 Lux light intensity. After pre-culture, the *Nematocystis* adhering to the reactor walls and plastic plates was scraped off, and the remaining *Nematocystis* in the culture medium was collected using a 300-mesh filter and combined. 50 g of wet *Nematocystis* was taken and washed three times with brackish water (salinity 5‰). The washed *Nematocystis* was inoculated into 0.5 L of brackish water (salinity 5‰) in a 1 L Erlenmeyer flask and cultured at 40°C and 1000 Lux light intensity. After 3 days of culture, the salinity of the brackish water decreased from the initial 5‰ to 4‰.

[0054] Example 4

[0055] *Nematocystis* was pre-cultured for 10 days in artificial seawater with a salinity of 160‰ in 2 L glass Erlenmeyer flasks using SYSU-Lvsan-1 culture medium at 40℃ and a light intensity of 5000 Lux. After pre-culture, the *Nematocystis* adhering to the walls of the Erlenmeyer flasks was scraped off, and the remaining *Nematocystis* in the culture medium was collected by filtering through a 300-mesh sieve and combined with the culture medium. 3 g of wet *Nematocystis* was taken and washed three times with brackish water (salinity 80‰). The washed *Nematocystis* was then inoculated into 3 L of brackish water (salinity 80‰) and cultured in a 5 L acrylic column photobioreactor at 20℃ and a light intensity of 5000 Lux. After 30 days of culture, the salinity of the brackish water decreased from the initial 80‰ to 73‰.

[0056] Example 5

[0057] *Nematocystis* was pre-cultured for one day in artificial seawater with a salinity of 50‰. The culture container was a 5 L plastic basin containing two nylon plates. The culture medium was F / 2, and the culture conditions were 30℃ and 2000 Lux light intensity. After pre-culture, the *Nematocystis* attached to the nylon plates was scraped off, and the remaining *Nematocystis* in the culture medium was collected using a 300-mesh filter and combined. 10 g of wet *Nematocystis* was taken and washed three times with brackish water (salinity 35‰). The washed *Nematocystis* was inoculated into 2 L of brackish water (salinity 35‰) and cultured in a 3 L acrylic tubular photobioreactor at 25℃ and 2000 Lux light intensity. After 30 days of culture, the salinity of the brackish water decreased from the initial 35‰ to 30‰.

[0058] Example 6

[0059] *Nematocystis* was pre-cultured for 30 days in artificial seawater with a salinity of 30‰. The culture container was a 20 L nylon tank with eight acrylic plates inserted. The culture medium was Zarrouk, and the culture conditions were 25℃ and 3000 Lux light intensity. After pre-culture, the *Nematocystis* attached to the acrylic plates was scraped off, and the remaining *Nematocystis* in the culture medium was collected using a 300-mesh filter and combined. 100 g of wet *Nematocystis* was taken and washed three times with brackish water (salinity 30‰). The washed *Nematocystis* was inoculated into 1 L of brackish water (salinity 30‰) in a 2 L plastic basin and cultured at 25℃ and 3000 Lux light intensity. After one day of culture, the salinity of the brackish water decreased from the initial 30‰ to 28‰.

[0060] Example 7

[0061] In Example 2, brackish water (salinity 18‰) was desalinated with 10 g of wet weight Nematoda for 14 days. The biomass of Nematoda increased to 20 g wet weight. 10 g of wet weight Nematoda was removed, and the remaining Nematoda was cultured at 25°C and 3000 Lux light intensity for another 18 days. The salinity of the brackish water decreased from 18‰ to 16‰.

[0062] Example 8

[0063] After separating the brackish water (salinity 18‰) that had been desalinated for 14 days with 10 g of wet-replenished Nematocystis in Example 2 from the Nematocystis, 10 g of wet-replenished Nematocystis was added to the treated brackish water. After culturing for another 18 days at 25°C and 3000 Lux light intensity, the salinity of the brackish water decreased from 18‰ to 15‰.

[0064] Example 9

[0065] After separating the brackish water (salinity 18‰) that had been desalinated for 14 days with 10 g of wet-weight Nematoda from the Nematoda in Example 2, 20 g of wet-weight Nematoda was obtained. 4 L of new brackish water was added to the Nematoda treated with brackish water, and the mixture was cultured for another 10 days at 25°C and a light intensity of 3000 Lux. The salinity of the brackish water decreased from the initial 20‰ to 16‰.

[0066] The above detailed description is a specific description of the embodiments of the present invention. These embodiments are not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included in the patent scope of this case.

Claims

1. Nematocystis ( Nodosilinea sp.) MH-2, characterized in that, The depositary institution is the China Center for Type Culture Collection, and the accession number is CCTCC NO: M 2026092.

2. The application of the Nematocystis MH-2 as described in claim 1 in brackish water desalination treatment.

3. A method for desalinating brackish water using Nematocystis aeruginosa, characterized in that, Includes the following steps: (1) Pre-culture: The *Nematocystis* described in claim 1 is inoculated into fresh water or artificial seawater for pre-culture; (2) Collection and washing: After the pre-culture is completed, collect the Nematocystis and wash the Nematocystis with brackish water to be treated; (3) Inoculation: Inoculate the washed Nematocystis into the brackish water to be treated; (4) Desalination treatment: Cultivate under light conditions and use Nematoda to reduce the salinity of brackish water.

4. The method according to claim 3, characterized in that, The pre-culture time in step (1) is 1-30 days, and the culture medium is F / 2 medium, Zarrouk medium, BG11 medium or modified medium SYSU-Lvsan-1. The culture conditions are: salinity 0-160‰, temperature 20-40℃, and light intensity 1000-5000 Lux.

5. The method according to claim 4, characterized in that, The formulation of the SYSU-Lvsan-1 medium is: urea 53.00 mg / L, NaH2PO4·H2O 5.65 mg / L.

6. The method according to claim 3, characterized in that, The inoculation ratio described in step (3) is 1-100 g wet Nematocystis thunbergii / L brackish water.

7. The method according to claim 3, characterized in that, The cultivation conditions described in step (4) are: temperature 20-40℃, light intensity 1000-5000 Lux.

8. The method according to claim 3, characterized in that, The cultivation device for the *Nematocystis* includes an open culture container or a closed photobioreactor; the open culture container includes a conical flask, a basin, or a tank; the closed photobioreactor includes an intermittent photobioreactor or a continuous flow photobioreactor.

9. The method according to claim 8, characterized in that, The culture device also includes a culture container with a support material selected from glass, plastic, nylon, or acrylic.

10. The method according to claim 3, characterized in that, In step (2), after the pre-culture is completed, the Nematocystis is collected by filtration and / or scraping.

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