Method for rapidly optimizing solid calcareous sand bacteria based on exopolysaccharide content detection

Abstract

The invention discloses a method for rapidly optimizing calcareous sand fixing bacteria based on extracellular polysaccharide content detection, which is used for optimizing bacterial strains with excellent sand fixing performance and rapidly providing microbial resources for reducing coral island soil erosion. The method provided by the invention can shorten the subsequent inspection on the calcareous sand consolidation capacity of the strain, can judge the sand consolidation capacity of the strain only by accurately measuring the exopolysaccharide content of the purified strain, can greatly save the time cost, provides a large number of strain resources for sand consolidation and erosion prevention of the coral island, and has a wide application prospect. The method has the advantages of simple operation, low cost, easy popularization and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial function screening, and is applicable to calcareous sand substrates. Specifically, it relates to a method for rapidly selecting bacteria for fixing calcareous sand based on the detection of extracellular polysaccharide content. Background Art

[0002] Most of the South China Sea Islands are coral islands, including 49 grey sand islands that can emerge above the water surface even at high tide. These islands are mostly composed of calcareous sand, in which calcium carbonate accounts for more than 95%, and the structure is loose and not conducive to the natural colonization of vegetation. The vegetation on tropical coral islands is extremely vulnerable to degradation and is extremely challenging to restore once damaged. Therefore, these coral islands are also regarded as the "deserts" in the ocean. This predicament is caused by a variety of unique environmental attributes, including the scarcity of soil clay and essential nutrients, increased salinity and alkalinity, intense high temperature and solar radiation, and recurring dry periods. Due to the lack of the ecological functions of vegetation, soil erosion is extremely likely to occur on coral islands. Therefore, preventing soil erosion is an urgent problem to be solved in this region.

[0003] Using soil crusts formed by microorganisms for sand fixation has increasingly emerged in the desertification control of arid and semi-arid regions. This method has the advantages of strong adaptability, low cost, and quick results. The microorganisms in desert crusts are not only participants in crust formation but also important components of the crust, and have the functions of enhancing soil aggregate stability, improving the moisture condition of the surface soil, and preventing soil erosion. Existing research has shown that the formation of soil crusts is closely related to microorganisms and the polysaccharide substances secreted by them. Bacteria play an important role in the initial stage of soil crust formation and secrete a large amount of extracellular polymers, namely extracellular polysaccharides, during their metabolic processes. Polysaccharide substances can cement soil particles into aggregates and play a binding role in the formation and stability of aggregates. At present, the microorganisms used for sand fixation mainly focus on the Cyanophyta, mainly including filamentous cyanobacteria such as Nostoc, Oscillatoria, Cylindrospermum, and Phormidium. Early research also found that some actinomycetes or Lysobacter can also be used for sand fixation to stabilize sand and gravel. It is reported that although many bacterial groups currently have the function of sand fixation, there is no relevant method for standardized selection. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for rapidly selecting bacteria for fixing calcareous sand based on the detection of extracellular polysaccharide content, and to select bacterial strains with excellent sand fixation performance to provide microbial resources for reducing soil erosion on coral islands.

[0005] In view of the lack of research on sand consolidation specifically for calcareous sand matrices at home and abroad, the present invention uses in situ calcareous sand and biological crust as research objects, separates and purifies microbial strains, and then combines the phenol-sulfuric acid method to determine the content of microbial extracellular polysaccharides based on the glucose standard curve, thereby screening the microbial strains with the highest extracellular polysaccharide content, see Table 1.

[0006] Based on the results of the extracellular polysaccharide content test, it was verified whether the calcium sand fixing function of these strains was positively correlated with their extracellular polysaccharide content. When the TSB culture solution of these pure bacteria was evenly sprayed on sterile calcium sand with a particle size of less than 1.25mm and greater than 0.20mm, after 60 days of cultivation, it was found that it could effectively promote the formation of crusts. The thickest crust formed was SCSIO17096, which was 4.4±1.4mm, and the thinnest crust was SCSIO17020, which was 1.3±0.7mm. The formed lumps were very easy to break. The crusts formed by the remaining strains were basically ranked according to the extracellular polysaccharide content, as shown in Table 2. Through the verification test, we found that the level of extracellular polysaccharide content determines its ability to fix calcium sand.

[0007] Therefore, the first object of the present invention is to provide a method for quickly selecting calcium-fixing sand bacteria based on the detection of extracellular polysaccharide content, and to select strains with high calcium-fixing sand ability by detecting the content of microbial extracellular polysaccharides.

[0008] Preferably, the method comprises the following steps: purifying and culturing the microorganisms of the collected target sample, then fermenting and culturing the pure bacteria in a fixed period, and detecting the extracellular polysaccharide content; based on the detection results of the extracellular polysaccharide content, selecting strains with high calcium sand fixing ability, and finally determining their classification information by high-throughput sequencing.

[0009] Preferably, the calcareous sand fixing ability of the strain is proportional to the extracellular polysaccharide content.

[0010] Preferably, the phenol-sulfuric acid method is used to determine the content of extracellular polysaccharides.

[0011] The second object of the present invention is to provide an application of the method in optimizing calcareous sand-fixing bacterial species resources and in coral island sand fixation and erosion prevention.

[0012] The method provided by the present invention can save a large number of subsequent functional verification processes. It only needs to accurately measure the extracellular polysaccharide content of the purified strain to determine the quality of its sand fixation ability, which can save a lot of time and cost, and provide a large number of bacterial resources for coral island sand fixation and erosion prevention. It has the advantages of simple operation, low cost, and easy promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1It is the research results of the characteristics of 8 pure strains and the blank group of calcareous sand. Specific Embodiments

[0014] The following examples are further illustrations of the present invention rather than limitations thereof.

[0015] Example 1: Determination of the extracellular polysaccharide content of each type of strain that has been isolated and purified

[0016] Randomly select 8 strains of bacteria from the isolated and purified strain bank for the determination of extracellular polysaccharide content. The ethanol precipitation method and the phenol-sulfuric acid method are used to determine the polysaccharide content of the extracellular polysaccharide sample. The specific operations are as follows:

[0017] Inoculate 8 strains of bacterial strains into TSB medium respectively. The medium components are tryptone 17.0 g / L, soy peptone 3.0 g / L, glucose 2.5 g / L, sodium chloride 5.0 g / L, K 2 HPO 4 2.5 g / L, the solvent is water, pH 7.3 ± 0.2. Its preparation method is to mix the above components evenly according to their contents, adjust the pH value, and then sterilize to obtain. Under the conditions of 30 °C and 180 r / min, culture for 48 h. Centrifuge the culture solution at 11603 g for 20 min to remove the bacteria, and then filter the supernatant through a 0.22 μm filter membrane to fully remove the residual microbial cells. Then pour the filtrate into a test tube, add 4 times the volume of 95% ethanol aqueous solution by volume, and precipitate overnight at 4 °C. Then centrifuge the mixture at 2057 g for 20 min, remove the supernatant, and collect the precipitate. The obtained precipitate is washed successively with acetone and absolute ethanol, and then 4 mL of 80% trichloroacetic acid by volume is added to the washed precipitate to remove proteins. Finally, remove the liquid, place the precipitate in an oven at 60 °C and dry for 30 h to obtain the extracellular polysaccharide sample.

[0018] Prepare a monosaccharide standard curve: Accurately weigh 10 mg of glucose (analytical pure) in a 50 mL beaker, dissolve it fully with deionized water, pour the aqueous solution into a 250 mL volumetric flask, and make up the volume with deionized water. Respectively pipette 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL into test tubes, and make up the volume to 1.0 mL with water in each case. Then add 0.5 mL of 6% phenol and 2.5 mL of concentrated sulfuric acid (analytical pure, 95.5%), let it stand for 10 min, shake well, and measure the absorbance at 490 nm with a spectrophotometer after standing at room temperature for 20 min. Use 1.0 mL of deionized water for the same color development operation as the blank control. The abscissa is the glucose mass, and the ordinate is the light absorption value, and draw the standard curve.

[0019] The extracellular polysaccharide samples of each prepared strain were redissolved in deionized water with the same volume as the original strain culture solution to make a solution. Pipette 0.8 mL from the sample solution into a test tube, and then add water to make up to 1.0 mL. Then add 0.5 mL of 6% phenol and 2.5 mL of concentrated sulfuric acid, let it stand for 10 min, shake well, and measure the absorbance at 490 nm using a spectrophotometer after standing at room temperature for 20 min. Use 1.0 mL of deionized water with the same color development operation as the blank control, measure the absorbance, and calculate the polysaccharide content according to the standard curve. After 48 hours of culture, the extracellular polysaccharide contents of 8 strains are shown in Table 1. The maximum value is SCSIO17096, which is 0.0403 mg / mL, and the minimum value is SCSIO17020, which is 0.0131 mg / mL.

[0020] Table 1 Detection results of extracellular polysaccharide contents of strains

[0021]

[0022]

[0023] Example 2: Determination of the ability of strains to fix calcareous sand

[0024] The coral sand was successively passed through 16-mesh (sieve hole 1.25 mm) and 80-mesh (sieve hole 0.20 mm) sieves to obtain coral sand with a particle size less than 1.25 mm and greater than 0.20 mm, which was respectively filled into different glass culture dishes, and 3 replicates were made for each sand quality specification, and then sterilized by high temperature. The 8 strains were respectively inoculated into TSB medium (same as Example 1), and after shaking culture at 30 °C and 180 r / min for 48 h, the bacterial solution was filled into a sterile small sprayer and evenly sprayed on the calcareous sand, with the sterilized TSB medium as the blank control. After culturing at 30 °C for 60 days, the thickness of the crust was measured. The detection results show that the bacterial strains producing extracellular polysaccharides can all effectively promote the formation of the crust. When the culture solution was evenly sprayed on the sterile calcareous sand with a particle size less than 1.25 mm and greater than 0.20 mm, the thickest crust was formed by SCSIO17096, which was 4.4 ± 1.4 mm, and the thinnest crust was formed by SCSIO17020, which was 1.3 ± 0.7 mm, and the formed lumps were extremely easy to break. The crusts formed by the remaining strains were basically sorted according to the level of extracellular polysaccharide content ( Figure 1 and Table 2). Thus, it can be seen that the method provided by the present invention can shorten the subsequent inspection of the function of strains to fix calcareous sand, and it can be basically determined that the higher the extracellular polysaccharide content, the stronger its ability to fix calcareous sand. Using the method of this invention can also quickly provide a large number of strain resources for sand fixation and erosion prevention on coral islands, and provide a good physical basis for the natural succession of coral islands.

[0025] Research Results on Sand Fixation Characteristics of Each Strain in Table 2

[0026]

[0027]

[0028] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for rapid selection of calcium-fixing sand bacteria based on exopolysaccharide content detection, characterized in that: By detecting the content of microbial extracellular polysaccharides, strains with high calcium sand fixing ability can be selected.

2. The method according to claim 1, characterized in that The method comprises the following steps: purifying and culturing the collected target samples of microorganisms, then fermenting and culturing the pure bacteria for a fixed period, and detecting the extracellular polysaccharide content; selecting strains with high calcium sand fixation ability based on the detection results of the extracellular polysaccharide content, and finally determining their classification information by high-throughput sequencing.

3. The method according to claim 2, characterized in that The calcium sand fixing ability of the strain is directly proportional to the extracellular polysaccharide content.

4. The method according to claim 2, characterized in that: The phenol-sulfuric acid method was used to determine the content of extracellular polysaccharides.

5. Application of the method according to any one of claims 1 to 4 in optimizing calcareous sand consolidation bacterial resources and / or coral island sand consolidation and erosion prevention.

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