CN115820422A - High-yield exopolysaccharide microalgae and application thereof in improving saline-alkali soil - Google Patents

Abstract

The invention provides a high-yield exopolysaccharide microalgae and application thereof in improving saline-alkali soil, wherein the high-yield exopolysaccharide microalgae is preserved in China Center for Type Culture Collection (CCTCC) with a preservation date of 2022 years, 6 months and 14 days and a preservation number of CCTCC NO: m2022884. The high-yield exopolysaccharide microalgae can obviously reduce the pH and the conductivity of saline-alkali soil, improve the content of organic matters, total sugar and enzyme activity of saline-alkali soil, and can be used for improving the saline-alkali soil.

Description

A high-yield exopolysaccharide microalgae and its application in improving saline-alkali soil

technical field

The invention belongs to the field of soil improvement, and in particular relates to a high-yield extracellular polysaccharide microalgae and its application in improving saline-alkali soil.

Background technique

Saline-alkali soil is a typical degraded soil. There are more than 1.125 billion hectares of saline-alkali soil distributed around the world, and about 800 million hectares of available arable land are affected by salinization. The area of saline-alkali land in China is about 99 million hectares, mainly distributed in the Northeast Plain, Northwest arid and semi-arid areas, Huanghuaihai Plain and eastern coastal areas. The most notable characteristic of saline-alkali soil is that its pH value is higher than that of normal soil, and soil salinity is a major abiotic stress factor that deteriorates soil quality, resulting in inhibited plant growth. Salt stress also affects soil biological properties by inhibiting enzyme activities and reducing the abundance and diversity of microbial communities.

At present, there are various methods of improving saline-alkali soil, including physical, chemical, biological and engineering improvement, to improve soil quality and crop yield. Chinese patent (application number: 202111343482.7) uses citric acid, amino acid powder, ammonium nitrate, urea phosphate, mineral source fulvic acid, enzymatic pollen and water to improve saline-alkali soil, but multiple applications are required during the implementation process, which increases the cost and Chemical additions always have two sides to soil quality. Chinese patent (application number: 202120710554.6) has developed a fast recyclable improved device for salt elution in saline-alkali soil, but the input cost and regular maintenance cost of these devices are relatively high, making sustainable development difficult.

Biological improvement methods have the advantages of resource saving, eco-friendliness, and environmental economy, and have attracted more and more attention from all parties. Microalgae are important producers in natural ecosystems, and play an indispensable role in the transformation of soil elements, especially the carbon cycle, which is conducive to increasing soil fertility. Exopolysaccharides secreted by microalgae can bind soil particles to form a three-dimensional extracellular polymeric matrix, which plays an important role in corrosion resistance, water retention, and protection against harmful environmental stress. Therefore, finding ways to use microalgae to improve saline-alkali soil has important practical significance for exerting its ecological functions and improving the quality of saline-alkali soil. Chinese patent (application number: 201910775218.7) provides a high-efficiency algae active saline soil treatment technology; Chinese patent (application number: 202010231785.9) provides a plant nutrition composition with microalgae as raw material and its preparation method. The above inventions are all compound nutrients prepared by mixing various microalgae, and some of them need to be applied multiple times during the application process, the dosage is large, and the investment and maintenance costs are high, so they face certain difficulties in the treatment of large-area saline-alkali land.

Contents of the invention

Aiming at the problems existing in the prior art, the present invention provides a high-yielding exopolysaccharide microalgae and its application in improving saline-alkali soil. Alkaline earth organic matter, total sugar content and enzyme activity.

The present invention is realized through the following technical solutions:

A high-yield extracellular polysaccharide microalgae was screened from the surface layer of a saline-alkali soil in the Guanzhong Plain, named Scenedesmussp. YJ-2, and was preserved in the China Center for Type Culture Collection (Wuhan University Collection Center) on June 14, 2022, with the accession number It is CCTCC NO: M2022884.

The characteristics of YJ-2 are: in BG-11 liquid medium, it can reach its logarithmic growth phase after 3 days of artificial climate culture; through microscope observation, YJ-2 cells are usually in the form of 2, 4 or 8 superimposed on each other, Palisade-like or quadrangular in plan, cell walls are smooth, with spines, teeth or raised lines.

The 18S rDNA gene sequence characteristics of YJ-2 are: the 18S rDNA sequence length is 653bp, and the 18S rDNA sequence was compared with the GenBank database by BLAST, and it was found that this algal strain has a higher Homology: Combined with the observation results of its morphology and structure, the algae strain was identified as Scenedesmus sp., named Scenedesmus sp. YJ-2 (Scenedesmus sp. YJ-2). The 18S rDNA sequence is shown as SEQ ID NO: 1, specifically:

cttccgtaggggaacctgcggaaggatcattgaatatgcaaaccacaacacgcactctttatttgtgttcgacgttaggtcaacacgcgcaagcgtgtggcctactaacctacacaccattgaccaaccatttatcaaa ccaaactctgaagctttggctgccgttaaccggcagttctaacaaagaacaactctcaacaacggatatct tggctctcgcaacgatgaagaacgcagcgaaatgcgatacgtagtgtgaattgcagaattccgtgaacc atcgaatctttgaacgcatattgcgctcgactcctcggagaagagcatgtctgcctcagcgtcggtttacac cctcacccctcttccttaacaggaggcgcctgtcgtgcttgcttaagccggcagcaggggtggatctggctc tcccaatcggattcactctggttgggttggctgaagcacagaggcttaaactgggacccaattcgggctca actggataggtagcaacaccctcgggtgcctacacgaagttgtgtctgaggacctggttaggagccaag caggaaacgcgtctctggcgcgtactctgtattcgacctgagctcaggcaaggctacccgctgaacttaa gcatatcaataagccggagg。

The application of the above-mentioned Scenedesmus YJ-2 in improving saline-alkali soil. The specific application is to inoculate the YJ-2 algae liquid in the logarithmic growth phase to the surface of saline-alkali soil, grow and form a layer of uniform algae crust, and then improve the saline-alkali soil.

The method for inoculating Scenedesmus YJ-2 to improve saline-alkali soil comprises the steps:

(1) Inoculate the purified Scenedesmus YJ-2 into a sterile liquid medium, and culture it to the logarithmic phase under light to obtain a highly active Scenedesmus YJ-2 algae liquid.

(2) Evenly spray and inoculate Scenedesmus YJ-2 algae liquid on the surface of saline-alkali soil, place the inoculated saline-alkali soil in an artificial climate incubator for cultivation, replenish water regularly, and form microalgae biocrusts that grow evenly on the surface of saline-alkali soil .

In the above method, the sterile liquid medium used in step (1) is BG-11 medium.

In the above method, the total chlorophyll content in the Scenedesmus YJ-2 liquid in step (2) is 2-5 μg/mL.

In the above method, the Scenedesmus YJ-2 algae solution in step (2) is uniformly inoculated on the surface of the saline-alkali soil at a rate of 0.24±0.06 μg (total chlorophyll)/cm ² .

In the above method, the saline-alkali soil in step (2) is mild to moderate saline-alkali soil, and the soil soluble salt content is 1.02-3.82g/kg.

In the above method, the cultivation conditions in the artificial climate incubator in step (2) are as follows: temperature 25°C, light intensity 120 μEm ^-2 s ^-1 , light/dark 16/8h, and water should be supplemented every day according to the change of soil moisture content, so that Its moisture content is kept at 20-25%.

In the above method, the culture time after inoculation of Scenedesmus YJ-2 in saline-alkali soil is 15-60 days.

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

The present invention obtains a high-yielding exopolysaccharide microalgae from saline-alkali land through a reasonable screening process, named Scenedesmus YJ-2, which belongs to indigenous microalgae, and the microalgae has high exopolysaccharide production, and is applied to saline-alkali soil after a large number of propagation Improvement can improve the quality of saline-alkali soil, and can achieve a good effect of improving saline-alkali soil without compounding with other algae, so the input cost is very low, and microalgae has good environmental adaptability.

In the present invention, the YJ-2 is inoculated in the saline-alkali soil through the environment control of good growth conditions, and a stable algae crust can be formed on the surface of the saline-alkali soil in a short period of time, and then the saline-alkali soil is improved through the metabolic activity of the microalgae. After the invention is inoculated with YJ-2, the pH and electrical conductivity of the saline-alkali soil show a trend of obvious decrease, and the organic matter, total sugar content and soil enzyme activity show a trend of increase, indicating that YJ-2 can effectively improve the quality of saline-alkali soil and has potential application prospects , to provide a way to improve saline-alkali soil.

Furthermore, the dosage of the microalgae liquid of the present invention is low, which can reduce the cost and is beneficial to the promotion of large-area use.

Description of drawings

Figure 1 shows the morphological characteristics of Scenedesmus YJ-2 under microscope observation.

Figure 2 is the molecular biology phylogenetic tree of Scenedesmus YJ-2.

Figure 3 shows the algal crusts formed on the surface of saline-alkali soil 15 days after Scenedesmus YJ-2 was inoculated.

Figure 4 is the dynamic change curve of microalgae biomass in saline-alkali soil after inoculation with Scenedesmus YJ-2.

Detailed ways

In order to further understand the present invention, the present invention will be described below in conjunction with the examples. These descriptions are only to further explain the features and advantages of the present invention, and are not intended to limit the claims of the present invention.

Example 1: Screening of indigenous microalgae in saline-alkali soil

Collect surface soil (0-1cm) with microalgae in natural mild to moderate saline-alkali land, place the surface soil sample in a sterilized 250mL transparent Erlenmeyer flask, add sterilized BG-11 liquid medium to the surface soil sample Most of them were submerged but not completely submerged, and then placed in an artificial climate incubator at 25°C, light intensity 120μEm ^-2 s ^-1 , light/dark 16/8h for enrichment culture. After the surface of the surface soil sample turns green obviously, pick a little sample and transfer it to a new BG-11 liquid medium for enrichment culture again, and then use the plate coating method to dilute the algae liquid onto the solid BG-11 medium Separation and purification are carried out to obtain a single algal cell colony. Pick a number of single-cell colonies, continue to expand the culture with liquid medium, and observe the shape of algal cells with a microscope until a single pure algae strain is obtained. Several pure algae strains obtained by screening were preserved in solid BG-11 medium for later use.

Example 2: Identification of the ability of microalgae to produce exopolysaccharides

(1) Qualitative detection of exopolysaccharides produced by microalgae

Add 100mL sterilized BG-11 liquid medium into a 250mL transparent Erlenmeyer flask, inoculate several algae strains stored on solid BG-11 medium into BG-11 liquid medium, place at 25°C, light intensity 120μEm ^-2 s ^-1 , light/dark 16/8h artificial climate incubator for enrichment culture for 3 days, fully shake the medium every 2 hours during the light period, to promote the reproduction of microalgae and avoid the aggregation of algal cells. After culturing for 3 days, collect the microalgae culture solution, add 2 times the volume of absolute ethanol solution, let it stand for 2 hours, then centrifuge at 8000rpm for 15 minutes, dissolve the precipitate in 10mL deionized water, add 1mL DNS reagent, and react in a boiling water bath for 5 minutes. After cooling Observe whether the solution shows reddish brown, if it shows reddish brown, it means that the microalgae secretes exopolysaccharide. Compare the shades of reddish-brown in different microalgae reaction solutions, choose the darkest strain and name it YJ-2.

(2) Determination of exopolysaccharide content

The content of exopolysaccharides was determined by the phenol-sulfuric acid method. Accurately weigh 50 mg of glucose dried at 105°C to constant weight, dissolve in distilled water, set the volume to a 50 mL volumetric flask, prepare a 0.1 mg/mL standard solution, and use distilled water to prepare 0, 0.02, 0.04, 0.06, 0.08, 0.1mg/L. Take 1mL of standard solutions with different concentration gradients, add 1mL of 5% phenol solution respectively, then quickly add 5mL of concentrated sulfuric acid, let stand for 10min, and shake well. After standing at room temperature for 30 minutes, measure the absorbance at 490 nm, take the glucose content as the abscissa, and the absorbance value as the ordinate, and make a standard curve.

The microalgae YJ-2 obtained in this embodiment had an exopolysaccharide yield of 312.36 ± 25.63 mg/L under conventional culture conditions.

Example 3: Identification of YJ-2

The morphological characteristics and taxonomic status of YJ-2 were observed and identified according to the following methods.

(1) Observed under a microscope, YJ-2 cells are usually in the form of 2, 4 or 8 superimposed on each other, arranged in a palisade or quadrangular shape on the plane, with smooth cell walls, spines, teeth or raised lines, as shown in Figure 1 Show.

(2) Identification of taxonomic status of YJ-2: microalgae 18S rDNA gene sequencing included algal strain DNA extraction, 18S rDNA in vitro PCR amplification (using 18S universal primers ITS1[5'-TCCGTAGGTGAACCTGCGG-3'] and ITS4[5' -TCCTCCGCTTATTGATATGC-3']) A gene fragment of 653 bp was obtained. The sequence results obtained by sequencing were brought into the NCBI database for BLAST gene sequence comparison. After analysis by MEGA software, they were compared with the 18S rDNA sequences of similar strains in the database, and the 18S rDNA phylogenetic tree of the strains was established, as shown in Figure 2 . The results showed that microalgae YJ-2 had a high homology with Scenedesmussp., with a similarity of 78%. Combined with the results of microscope morphology observation, the algae strain was identified as Scenedesmus sp, serial number OM964574.

Example 4: Growth status of YJ-2 in saline-alkali soil and changes in properties of saline-alkali soil after inoculation of YJ-2

Inoculate the YJ-2 algae liquid evenly on the surface of saline-alkali soil at 0.28 μg (total chlorophyll)/cm ² , cultivate it in an artificial climate incubator, replenish water every day to make up for the lost water, and keep the water content at 20-25 %, 15d can form a layer of obvious algae crust (as shown in Figure 3). The dynamic changes of microalgae biomass in saline-alkaline soil were measured at 15, 30, 45 and 60 days, and the pH, electrical conductivity, organic matter, total sugar content and enzyme activity of saline-alkali soil were measured at 60 days.

Microalgae biomass determination method in saline-alkali soil: take algal crust soil per unit area (1cm diameter) into a 10mL centrifuge tube, add 5mL absolute ethanol, shake well, then extract in dark for 24 hours, shake again 3 times during the period, and Chlorophyll was extracted from saline-alkali soil, centrifuged at 8000rpm for 15min after extraction, and the absorbance of the supernatant was measured at 665 and 652nm to calculate the chlorophyll content. The dynamic change curve of microalgal biomass in saline-alkali soil after inoculation of YJ-2 is shown in Figure 4. The chlorophyll content in algal crusts gradually increased with the culture time, indicating that YJ-2 showed good adaptability to saline-alkali soil.

The pH, electrical conductivity, organic matter, total sugar content, and activities of catalase, sucrase and urease in saline-alkali soil before and after inoculation with microalgae YJ-2 were measured. The pH and conductivity of saline-alkali soil were measured by pH meter and conductivity meter respectively, the organic matter was measured by potassium dichromate volumetric method, the catalase was measured by potassium permanganate titration method, and the sucrase was measured by 3,5-dinitro water Salicylic acid colorimetric method was used for determination of urease, sodium phenate-sodium hypochlorite colorimetric method was used for determination of urease, and total sugar was determined by phenol-sulfuric acid method. After inoculating YJ-2 and culturing for 60 days, the changes in some properties of the saline-alkali soil are shown in Table 1.

Table 1 Changes of pH, electrical conductivity, organic matter, enzyme activity and total sugar content in saline-alkali soil after inoculation of YJ-2 for 60 days

It can be seen from Table 1 that compared with the non-inoculated YJ-2, the pH and electrical conductivity of the saline-alkali soil decreased, and the organic matter, total sugar content and enzyme activity in the soil all showed varying degrees of increase, indicating that the inoculation of YJ-2 can improve the quality of saline-alkali soil .

Claims (10)

1. A high-yield exopolysaccharide microalgae is characterized in that the high-yield exopolysaccharide microalgae belongs to Scenedesmus sp, is preserved in China center for type culture Collection with a preservation date of 2022 years, 6 months and 14 days and a preservation number of CCTCC NO: m2022884.

2. The exopolysaccharide-producing microalgae of claim 1 wherein the DNA sequence of said exopolysaccharide-producing microalgae is as set forth in SEQ ID NO:1 is shown.

3. Use of the high exopolysaccharide microalgae according to any of claims 1-2 for improving saline-alkali soil.

4. The use according to claim 3, comprising:

(1) Inoculating the purified microalgae with high extracellular polysaccharide yield to a sterile liquid culture medium, and performing illumination culture until logarithmic phase to obtain microalgae solution;

(2) And uniformly spraying microalgae solution on the surface of saline-alkali soil, inoculating saline-alkali soil, culturing in artificial climate, and periodically supplementing water to form microalgae biological crust uniformly growing on the surface of the saline-alkali soil.

5. The use as claimed in claim 4, wherein in step (1), the sterile liquid medium is BG-11 medium.

6. The use of claim 4, wherein in the step (2), the total chlorophyll content in the microalgae suspension is 2-5 μ g/mL.

7. The use of claim 6, wherein the microalgae juice is 0.24 ± 0.06 μ g/cm calculated as chlorophyll ² Inoculating the strain on the surface of saline-alkali soil.

8. Use according to claim 4, characterized in that in step (2) the climatic conditions are: temperature 25 deg.C, light intensity 120 mu Em ^-2 s ^-1 The light/dark was 16/8h.

9. The use according to claim 4, wherein in step (2), the saline-alkali soil is periodically supplemented with water to maintain the water content of the saline-alkali soil at 20-25%.

10. The use according to claim 4, wherein the inoculated saline-alkali earth is cultured in a climatic environment for 15-60 days.

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