A kind of wall-adhering electroactive microalgae with salt stress response characteristics, biological preparation and application

Abstract

This invention relates to the field of microbiology and its application technology, specifically providing an adherent electroactive microalga with salt stress-responsive characteristics, biological agents, and applications. The microalga provided by this invention belongs to the phylum Cyanobacteria, specifically *Desertifilum salkalinema* UJS-X-1, with accession number CGMCC No. 46959. This algal strain can adhere to and grow on solid or semi-solid substrates to form biofilms, exhibiting stable extracellular electron transport capabilities, good salt and alkali tolerance, and a dose-response relationship between its electroactivity and salinity. It can significantly reduce the salinity and pH of saline-alkali soils, and under salt and alkali stress, it can promote rice growth, increase its biomass and chlorophyll content, and reduce malondialdehyde content. The algal strain provided by this invention can be used to prepare biological agents for saline-alkali land remediation, construct microalgal vegetation, serve as a salinity-responsive biological indicator, as an anode material for bio-batteries, and as a bio-fertilizer for crops in saline-alkali land, showing broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology and ecological restoration technology, specifically involving an electrochemically active microalga, and its response to stress factors such as salt, alkali and heavy metals under attached growth conditions and its ecological applications. Background Technology

[0002] Microalgae, as photosynthetic microorganisms, play a vital role in ecological restoration. Recent studies have revealed that certain cyanobacteria and microalgae possess light-dependent electroactivity, enabling them to transfer electrons generated during photosynthesis and respiration to the extracellular space. This electrochemical activity may be a crucial mechanism for the interaction between microalgae and their environment.

[0003] In arid, semi-arid, and degraded ecosystems, biocrusts are complexes formed by the combination of microorganisms such as cyanobacteria and soil particles, possessing significant ecological functions. Cyanobacteria, as key species in crust formation, are widely used in artificial crust construction and desertification control. However, existing algal species still face the following problems in practical applications: (1) slow growth, making it difficult to quickly form stable crusts; (2) poor adaptability to stressful environments such as salinity and alkalinity; and (3) lack of response mechanisms to environmental factors, making functional monitoring and regulation difficult.

[0004] Existing research on the electroactive properties of microalgae largely focuses on suspension culture systems. However, in practical ecological restoration applications, microalgae typically form biocrusts through adherent / attached growth. Therefore, studying the electroactive behavior of microalgae under adherent culture conditions and their response to environmental factors (such as salinity) has more direct reference value for guiding practical ecological restoration engineering. Currently, there are no reports on the electroactive properties of microalgae in adherent / open culture systems and their application in saline-alkali land remediation. Summary of the Invention

[0005] This invention provides an electroactive microalga with adherent growth characteristics, a biological agent, and its applications. This microalga not only rapidly forms a biological crust on the surface of a solid substrate, exhibiting certain salt tolerance and electrochemical activity, but its electrochemical activity also responds to salinity, reducing the salinity and alkalinity of saline-alkali soils, improving their pH, and increasing organic matter content. This microalga combines adherent growth, salt and alkali tolerance, electrochemical activity, and environmental responsiveness, making it widely applicable in saline-alkali land improvement, desertification control, promoting crop growth under salt and alkali stress, salinity detection, and electrochemical fields.

[0006] One objective of this invention is to provide an adherent electroactive microalga with salt stress response characteristics, characterized in that the electroactive microalga is Desert filamentous algae Desertifilum salkalinema UJS-X-1, belonging to the phylum Cyanobacteria, and is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 46959.

[0007] The application of the adherent electroactive microalgae as microalgal vegetation on bare ground surfaces.

[0008] Application of the adherent electroactive microalgae as electrochemical anodes in bio-batteries.

[0009] Application of the adherent electroactive microalgae in detecting salinity in soil or water.

[0010] Furthermore, the application of the aforementioned adherent electroactive microalgae in detecting soil or water salinity involves using a three-electrode cyclic voltammetry (CV) scanning method to determine the salinity of the soil or water to be tested by observing the correlation between the oxidation peak current of the electroactive microalgae and salinity.

[0011] Application of the adherent electroactive microalgae in saline-alkali land remediation.

[0012] The adherent electroactive microalgae are used as bio-fertilizers for crops in saline-alkali land.

[0013] A biological agent, characterized in that it contains the adherent electroactive microalgae as described in claim 1.

[0014] Furthermore, the biological agent also includes a biologically acceptable carrier.

[0015] Furthermore, the biological agent is a biosensor or biofertilizer for monitoring soil / water salinity.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) This invention has for the first time screened an electroactive microalga, *Desertifilum salkalinema* UJS-X-1, capable of stable growth under adherent conditions. This microalga belongs to the phylum Cyanobacteria and is a desert filamentous alga. The algal body is filamentous, unbranched, and enclosed by a transparent sheath, with a blue-green color. This desert filamentous alga can rapidly attach and grow on solid / semi-solid substrates, forming a uniform algal membrane or algal crust-like structure, with a biomass of up to 26.42 g / m². It has a certain degree of salt and alkali tolerance, and the algae can grow well in a salinity range of 0-15‰. Cyclic voltammetry (CV) analysis showed that the algae exhibited a characteristic oxidation peak in the potential range of -0.011 V to 0.015 V, indicating that it possesses extracellular electron transport capabilities. Furthermore, the electrochemical activity of this algae is closely related to salinity, showing that the peak current intensity of the oxidation peak decreases with increasing salinity, exhibiting a clear dose-response effect. This characteristic makes it an ideal biological indicator for monitoring salinity levels in soil or water.

[0018] Experiments on saline-alkali land remediation and rice cultivation have shown that the algae strain of this invention can significantly reduce the soluble salt content and pH value in saline-alkali soil, improving the soil's physical and chemical properties. Under saline-alkali stress, inoculation with the algae strain of this invention can significantly promote the growth of crops such as rice and increase leaf chlorophyll content, while reducing malondialdehyde content, effectively alleviating salt stress damage in rice and promoting rice growth, providing a novel green solution for the agricultural development and utilization of saline-alkali land.

[0019] (2) This algal strain has three characteristics: adherent growth, salt and alkali tolerance and electrochemical activity. It can be directly used to prepare biological agents, such as for the restoration of saline-alkali land, rapid construction of microalgal vegetation on bare ground, and for the improvement of saline-alkali land, soil carbon sequestration and stability enhancement. Attached Figure Description

[0020] Figure 1 This is a microscope image of the algal strain UJS-X-1 of this invention.

[0021] Figure 2 This is the phylogenetic tree of the algal strain UJS-X-1 of this invention.

[0022] Figure 3 The figure shows the effect of different sea salt concentrations on algal growth under adherent culture conditions. In the figure, BG11 is the normal BG11 medium with 0‰ sea salt; BG11-10 is the BG11 medium with 10‰ sea salt; BG11-15 is the BG11 medium with 15‰ sea salt; BG11-20 is the BG11 medium with 20‰ sea salt; and BG11-25 is the BG11 medium with 25‰ sea salt.

[0023] Figure 4This is a typical cyclic voltammetry (CV) curve of the algal strain of the present invention after adherent culture.

[0024] Figure 5 This is a superimposed CV curve of algal strains under different salinity treatments under adherent culture conditions. In the figure, BG11 is the normal BG11 medium with 0‰ sea salt; BG11-10 is the BG11 medium with 10‰ sea salt; BG11-15 is the BG11 medium with 15‰ sea salt; BG11-20 is the BG11 medium with 20‰ sea salt; and BG11-25 is the BG11 medium with 25‰ sea salt.

[0025] Figure 6 This is a graph showing the changes in soil salinity, pH, and organic matter in the application of the algal strain of this invention in the remediation of saline-alkali soil, where A: pH; B: salt content; C: organic matter content.

[0026] Figure 7 The image shows the effect of the algae strain of this invention on promoting the growth of rice seedlings under salt and alkali stress. In the image, A represents plant height; B represents chlorophyll a content in leaves; and C represents malondialdehyde content in leaves. Detailed Implementation

[0027] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental materials used in the following examples are all conventional reagents. Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0028] The basal culture medium used in this invention is BG11 medium. Unless otherwise specified, the culture conditions are: temperature 25℃, light intensity 130 μmol·m². -2-1 Light-dark ratio 24 h: 0.

[0029] Example 1: Isolation and purification of the electroactive microalgae Desert filamentous algae Desertifilum salkalinema UJS-X-1

[0030] Samples of crusted algae collected from a paddy field in Huai'an City, Jiangsu Province, were inoculated into BG11 liquid medium for enrichment culture. Single algal colonies were isolated on BG11 solid medium using the dilution plating method and the streak plating method. After multiple collections of single algal colonies and microscopic observation, a pure cultured algal strain was obtained and named UJS-X-1.

[0031] Under an optical microscope, the algal strain appeared filamentous, unbranched, and enclosed in a transparent sheath, with a bluish-green color. A photograph of its morphology is shown below. Figure 1As shown. The gene sequence of this algal species was obtained by 16S rRNA sequencing. Using the NCBI nucleic acid database, BLAST analysis was performed based on the extracted 16S rRNA gene sequence of the UJS-X-1 algal strain to construct a phylogenetic tree, as shown. Figure 2 As shown, the results indicate that the 16S rRNA gene sequence of the algal strain UJS-X-1 has the highest homology with Desert filamentum (Desertifilum salkalinema), and the strain was finally determined to belong to Desert filamentum and Cyanobacteria at the molecular level.

[0032] Biological Preservation Information: The isolated and purified desert filamentous algae strain *Desertifilum salkalinema* UJS-X-1 was deposited on March 12, 2026, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 46959. Testing confirmed that the strain is viable.

[0033] Example 2: Adherent growth characteristics

[0034] A certain number of cellulose membranes inoculated with the purified Desert filamentous algae strain *Desertifilum salkalinema* UJS-X-1 from Example 1 were placed on a sponge, and an appropriate amount of BG11 liquid culture medium was added to saturate them. The culture medium was then circulated using a circulation pump. The algae strain adhered to the substrate surface and grew, forming a uniform biofilm.

[0035] Example 3: Effects of different salinities on algal growth under adherent culture conditions

[0036] To simulate the complex salinity of real-world soils, BG11 culture solutions with sea salt concentrations of 0‰, 10‰, 15‰, 20‰, and 25‰ were prepared, with three replicates for each sea salt concentration. Algal cultures in the logarithmic growth phase were inoculated onto each filter membrane in equal volumes. The inoculated membranes were then placed on sponges, and the sponges were soaked with the prepared BG11 culture solutions containing different sea salt concentrations. The membranes were incubated statically for 7 days under standard light and temperature conditions. Algal patch expansion was observed daily, and biomass was measured. Biomass was determined using the dry weight per unit area method. The procedure involved taking a certain area of ​​cellulose filter membrane, scraping off all the algae, placing it on the weighed cellulose filter membrane, drying it, weighing it, and calculating the dry weight of the algae per unit area.

[0037] like Figure 3As shown, under adherent culture conditions, this algal strain grew well at a salinity of 0-15‰, exhibiting a high dry weight per unit area during the 7-day culture period. Growth was inhibited when the salinity increased to 20‰. These experimental results demonstrate that the desert filamentous algae *Desertifilum salkalinema* UJS-X-1 screened in this invention can tolerate salinity concentrations of 0-15‰.

[0038] Example 4: Electrochemical activity characterization of adherent algal strains

[0039] To simulate a real-world environment, the electrochemical activity assay was performed directly in the adherent culture system of BG11 medium, with no further treatment of the algae except under fixed experimental conditions.

[0040] A glassy carbon electrode with a diameter of 3 mm was used as the working electrode, an Ag / AgCl electrode as the reference electrode, and a platinum sheet electrode as the counter electrode. Saturated KCl was used as the electrolyte. Cyclic voltammetry (CV) scanning was performed using a three-electrode system to complete the electrochemical assay of the desert filamentous algae. The assay was conducted in an open system, requiring no strictly anaerobic conditions. The scanning parameters were: initial potential -0.8 V, high potential 0.8 V, low potential -0.8 V, termination potential 0.8 V, scan rate 0.01 V / s, settling time 2 s, and sensitivity 10. -5 A / V.

[0041] Test results are as follows Figure 5 As shown, compared with the blank control group without *Desmodium styracifolium*, the sample containing *Desmodium styracifolium* exhibited a distinct oxidation peak between -0.011 V and 0.015 V (vs. Ag / AgCl), with a peak current of approximately -9.544e-7 A. This indicates that the *Desmodium styracifolium* strain possesses extracellular electron transport capability under adherent growth conditions.

[0042] Example 5: Effect of different salinities on the electroactivity of algal strains under adherent culture conditions

[0043] Desert filamentous algae were cultured adherently to the culture wall to the logarithmic growth phase under salinity conditions of 0‰, 10‰, 15‰, 20‰, and 25‰, respectively, and electrochemical detection was performed. The results are as follows: Figure 6 As shown, the oxidation peak current of algal strains cultured at different salinities exhibited significant differences. With increasing salinity, the oxidation peak current decreased from -9.544e-7 A to 0 A. These results indicate that, to resist the external saline environment, the algae enhanced its own energy utilization, thereby reducing its interaction with the external environment.

[0044] Example 6: Application of the algal strain of the present invention in saline-alkali land remediation and rice cultivation

[0045] Coastal saline-alkali soil was collected from Rudong County, Jiangsu Province. The initial salt content was 3 g / kg, pH was 8.8, and organic matter was 8.49 g / kg. Three treatment groups were set up: (1) Control group (CK): the soil was not treated; (2) BG11 group: the soil was supplemented with BG11 culture medium with an equal volume of algal solution; (3) Algal solution treatment group (Algae): the algal solution of desert filamentous algae screened and purified in Example 1 was evenly sprayed onto the soil surface; each group had 3 replicates. The soil moisture content was made up to 60% and kept moist for a total of 90 days. The salinity, pH and organic matter content of the soil were measured every 30 days. After 90 days, rice seeds of variety Yanfeng 47 were planted in the soil of each treatment group. After growing in the greenhouse for 60 days, the plant height, chlorophyll content and malondialdehyde (MDA) content of the rice seedlings were measured.

[0046] Soil remediation effect Figure 6 As shown, after 90 days of treatment, the soil salinity of the algae treatment group (Algae) significantly decreased from 3.45 g / kg to 1.96 g / kg, the pH value decreased from 8.85 to 8.44, and the organic matter content significantly increased from 8.49 g / kg to 13.80 g / kg. In contrast, the salinity and pH value of the control group (CK) showed no significant changes. This indicates that the desert filamentous algae of the present invention can effectively reduce the salinity of saline-alkali land and increase the soil organic matter content.

[0047] Rice growth promotion test results as follows Figure 7 As shown, compared with the control group (CK), the rice seedlings in the algae treatment group (Algae) had a 28.2% increase in plant height. Simultaneously, the chlorophyll content of the rice leaves in the algae treatment group (Algae) increased by 38.7%, while the malondialdehyde (MDA) content decreased by 63.3%, indicating that this desert filamentous algae strain can effectively alleviate the oxidative damage caused by salt stress to rice and promote its growth in saline-alkali environments.

[0048] In summary, the electroactive microalgae *Desertifilum salkalinema* UJS-X-1 provided by this invention can adhere to and grow on solid or semi-solid substrates to form biofilms, exhibiting clear salinity tolerance and electrochemical activity under adherent culture conditions. Its growth and electrochemical activity are affected by salinity, indicating that this algal strain can sense and respond to changes in environmental salinity. Furthermore, this microalgae can effectively reduce salinity in saline-alkali lands and increase soil organic matter content, effectively alleviating oxidative damage to rice caused by salt stress and promoting rice growth in saline-alkali environments.

[0049] Utilizing these properties of the electroactive microalgae *Desertifilum salkalinema* UJS-X-1 provided by this invention, the *Desertifilum salkalinema* can be used for:

[0050] (1) Since the desert filamentous algae has the characteristics of adhering to the wall and salt tolerance, the desert filamentous algae can be inoculated on normal bare ground or saline-alkali bare ground to form artificial microalgae vegetation on saline-alkali bare ground, restore vegetation, enhance soil stability and reduce soil erosion.

[0051] (2) Based on the electrochemical activity characteristics of the desert filamentous algae, the filamentous algae can be used as an electrochemical anode material.

[0052] (3) Based on the response of the electrochemical activity of this desert filamentous algae to different salinities, it can be used to detect the salinity of the environment.

[0053] (4) Taking advantage of the desert filamentous algae’s ability to effectively reduce the salinity of saline-alkali land and increase the organic matter content of soil, this microalgae can be used for saline-alkali land remediation, reducing soil salinity and pH, and improving soil structure.

[0054] (5) Use the desert filamentous algae as a bio-fertilizer for planting crops (such as rice) in saline-alkali land, promote the growth of crops in saline-alkali land, and improve the tolerance and yield of crops such as rice under saline-alkali stress.

[0055] To facilitate the application of this desert filamentous algae in the aforementioned applications, the present invention also provides a method for preparing a biological agent from the desert filamentous algae described in this invention. In addition to containing the desert filamentous algae, the biological agent may also include biologically acceptable carriers, etc. Types of such biological agents include biosensors for monitoring soil / water salinity, biofertilizers, etc.

[0056] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A wall-adherent electroactive microalgae with salt stress response characteristics, characterized in that, The electroactive microalgae is Desert filamentous algae, *Desertifilum salkalinema* UJS-X-1, belonging to the phylum Cyanobacteria. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46959.

2. The application of the adherent electroactive microalgae of claim 1 as microalgal vegetation on bare ground surfaces.

3. The application of the adherent electroactive microalgae of claim 1 as an electrochemical anode in a bio-battery.

4. The application of the adherent electroactive microalgae of claim 1 in the detection of salinity in soil or water.

5. The application of the adherent electroactive microalgae according to claim 4 in the detection of soil or water salinity, characterized in that, The salinity of the soil or water to be tested was determined by using a three-electrode cyclic voltammetry (CV) scanning method and the correlation between the oxidation peak current of electroactive microalgae and salinity.

6. The application of the adherent electroactive microalgae of claim 1 in the remediation of saline-alkali land.

7. The application of the adherent electroactive microalgae of claim 1 as a bio-fertilizer for crops in saline-alkali land.

8. A biological agent, characterized in that, The biological agent contains the adherent electroactive microalgae as described in claim 1.

9. The biological agent according to claim 8, characterized in that, The biological agent also includes a biologically acceptable carrier.

10. The biological agent according to claim 8, characterized in that, The biological agent is a biosensor or biofertilizer for monitoring soil / water salinity.

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