Strain for producing exopolysaccharide and application of strain in sand fixation

Abstract

The invention belongs to the technical field of microorganisms, and discloses a strain for producing exopolysaccharide and application thereof in sand fixation. According to the invention, a strain for producing exopolysaccharides is separated and screened from rhizosphere soil of haloxylon ammodendron in Taklamakanensis, the strain is Paenibacillus taklamakanensis TRM82006, the strain secretes the exopolysaccharides, when the strain is applied to aeolian sandy soil, a biological membrane can be formed among soil particles, the motility of sand grains is reduced, wind erosion is effectively prevented, and after natural air drying, a microorganism solidification sand barrier can be formed, so that the effect of producing the exopolysaccharides is achieved. The soil structure can be improved, the soil moisture retention capability is improved, and the plant growth is promoted, so that the method has wide ecological and economic values. The invention provides a sand stabilization method which is environment-friendly, low in cost, quick in effect, simple and easy to implement and excellent in stability for desertification control and ecological restoration, and particularly shows huge application potential in extreme drought and barren environments.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms and relates to a polysaccharide-producing strain and application of the strain in sand fixation. BACKGROUND

[0002] Desertification, also known as desertification, is a land degradation process caused by the combined effects of natural factors and human activities, resulting in decreased land productivity and damaged ecological functions. It is mainly manifested as the expansion of sandy soil and the reduction of plant coverage. Desertification not only affects soil quality and reduces agricultural productivity, but also exacerbates environmental degradation and even leads to feedback effects on climate change. Currently, sand fixation technology systems include physical sand fixation, chemical sand fixation and biological sand fixation. Physical sand fixation uses engineering measures such as grass checkerboard, nylon netting and vegetation coverage to build mechanical sand barriers on the sand surface. Although it has cost advantages, its structure stability is easily damaged under the action of wind erosion and sandstorm disasters, and it requires continuous investment in maintenance resources. Chemical sand fixation technology uses adhesives to form a sand surface solidification layer, which can quickly improve the erosion resistance of sandy soil. However, the material cost is high, and the environmental compatibility and long-term stability of chemical agents still lack systematic verification. Biological sand fixation uses vegetation or microbial communities to fix flowing sand and improve the ecology of sandy areas. However, due to the dual constraints of water stress and soil barrenness in arid regions, the survival rate of plants is low and the forestation period is long, making it difficult to form an effective protection system in the short term.

[0003] Microbial sand fixation technology is a green sand fixation method that uses microorganisms as the core and achieves sand surface solidification and ecological restoration through biochemical mechanisms. In recent years, it has shown great potential in the governance of arid regions. Its core mechanisms include extracellular polysaccharide cementation and microbial-induced calcium carbonate precipitation (MICP), which have the advantages of "low cost, near nature and sustainability". Early research focused on the selection of sand-fixing microorganisms and the exploration of sand-fixing mechanisms, especially in the research of extracellular polysaccharide secretion. Although microbial sand fixation technology has been successful in laboratories and small-scale experiments, it still faces some challenges in large-scale applications, such as the survival ability of microorganisms in extreme environments and the long-term stability of sand fixation effects.

[0004] Therefore, it is of great significance for the governance and ecological restoration of desertification to isolate and screen strains that can survive in extreme environments and have good long-term stability of sand fixation effects. SUMMARY

[0005] To solve the above technical problems, the present application provides a strain producing exopolysaccharide and its application in sand fixation. The present application screens a strain producing exopolysaccharide from the rhizosphere soil of Haloxylon ammodendron in the Taklimakan Desert, and the strain is Paenibacillus taklamakanensis TRM82006. The strain secretes exopolysaccharide, and when applied to aeolian soil, it can form a biofilm between soil particles, reduce the mobility of sand particles, effectively prevent wind erosion, and form a microbial solidified sand barrier after natural drying. Therefore, it can improve soil structure, improve soil water retention capacity, and promote plant growth, and thus has wide ecological and economic value. The present application provides an environmentally friendly, low-cost, fast-acting, simple, stable and excellent sand fixation method for the treatment and ecological restoration of desertification, and has great application potential in extremely dry and barren environments.

[0006] In one aspect, the present application provides a strain producing exopolysaccharide, and the strain is Paenibacillus taklamakanensis TRM82006. The nucleotide sequence of 16S rRNA of the strain is shown in SEQ ID No: 1. The Paenibacillus taklamakanensis TRM82006 is a transparent protruding small colony in the early growth stage, and the colony is round, yellow, transparent protruding, gummy, sticky or more or less in the later stage. The strain TRM82006 is observed by scanning electron microscope, and the cell body is rod-shaped, and there is a large amount of sticky material outside the cell.

[0007] The strain producing exopolysaccharide Paenibacillus taklamakanensis TRM82006 of the present application is collected from the rhizosphere soil of Haloxylon ammodendron in the Taklimakan Desert. The preservation information is as follows:

[0008] Classification name: Paenibacillus taklamakanensis

[0009] Preservation time: May 21, 2025

[0010] Preservation unit: Guangdong Microbial Culture Collection Center

[0011] Address of the preservation unit: No. 59, Guangdong Microbial Culture Collection Center, 100, Martyrs Road, Guangzhou, Guangdong, China

[0012] Preservation number: GDMCC No. 66363

[0013] In another aspect, the present application claims the use of at least one of the strain, the fermentation broth of the strain, and the metabolite of the strain in sand fixation. The exopolysaccharide and viscous substance secreted by the strain can form stable biofilm, promote sand crust, enhance the binding effect between soil particles, and reduce the movement between soil particles, so that the wind erosion of sand is reduced after use.

[0014] Further, the preparation method of the fermentation broth comprises: inoculating Paenibacillus taklamakanensis TRM82006 in a 2% inoculation amount into Gao No. 1 medium, and performing shaking culture at 28-30°C and 120 r / min for 72 h. Preferably, the culture is performed until the OD 600 value is 2.0 to obtain the fermentation broth.

[0015] Further, the present application finds through a sand jar test that the liquid treatment group can form continuous biological crust within 10 days, and the average thickness and pressure-bearing capacity (hardness) of the crust of 75% of the liquid treatment group are significantly better than those of other groups. The centrifugal supernatant also has the effect of promoting the crust, but the effect is slightly weaker than that of the complete liquid.

[0016] Further, the present application finds through a desert field test that the pure water treatment group has sand collection peaks of 397.48 g and 459.80 g at the 20th day and the 40th day after the liquid treatment, indicating that the wind erosion is the most serious in the untreated state; the sand collection amount of the treatment group in which Haloxylon ammodendron is planted is between that of the pure water group and the liquid treatment group, indicating that the planting of Haloxylon ammodendron has a certain sand control effect; the sand collection amount of the 25% liquid treatment group slightly decreases compared with that of the pure water treatment group, but still reaches 182.08 g at the 40th day, and the sand control ability is limited; the sand collection amount of the 50% liquid treatment group rapidly decreases after reaching a peak of 303.02 g at the 40th day, and the sand control effect is certain; the sand collection amount of the 75% liquid treatment group is the least from the beginning to the end, and the sand collection amount at the 40th day is 127.21 g, which is significantly decreased compared with the sand collection amounts of the pure water treatment group and the positive control group, and the sand fixation ability is strong.

[0017] In another aspect, the present application claims a microbial agent, and the active ingredient comprises at least one of the Paenibacillus taklamakanensis TRM82006 strain, the fermentation broth of the strain, and the metabolite of the strain.

[0018] Further, the present application claims a microbial sand fixation method, and the above microbial agent is directly sprayed or diluted to 25-75% and then sprayed. The spraying amount is not less than 1 L / m 2 . Preferably, the microbial agent is diluted to 75% and then sprayed.

[0019] Compared with the prior art, the technical scheme provided by the present application has at least the following beneficial effects or advantages:

[0020] (1) The present application isolates and screens a strain producing exopolysaccharide from the rhizosphere soil of Haloxylon ammodendron in the Taklimakan Desert. The strain is Paenibacillus taklamakanensis TRM82006, which secretes exopolysaccharide. When applied to aeolian soil, the strain can form a stable biofilm between soil particles, reduce the mobility of sand particles, effectively prevent wind erosion, and form a microbial solidified sand barrier after natural drying. Therefore, the strain can improve soil structure, increase soil water retention capacity, and promote plant growth, and thus has wide ecological and economic value. Exopolysaccharide is a high molecular weight substance secreted by microorganisms, which has strong adhesion and can form a cemented structure in soil, increasing the cohesion between soil particles and thus enhancing the stability of sandy soil. The present application provides an environmentally friendly, low-cost, fast-acting, simple, stable sand-fixing method for the treatment and ecological restoration of desertification. Compared with chemical sand fixation and physical sand fixation, the sand-fixing method of the present application does not cause negative effects on the surrounding ecosystem, and does not require the addition of a large amount of chemical solidifying agent or the frequent replacement of physical barrier materials, thereby reducing the cost of materials and maintenance.

[0021] (2) The strain, the fermentation broth of the strain, and the centrifugal supernatant of the fermentation broth provided by the present application all have good sand-fixing effects. The present application found through sand jar tests that the bacterial liquid treatment group can form a continuous biological crust within 10 days, and the average thickness and pressure-bearing capacity (hardness) of the crust of 75% of the bacterial liquid treatment group are significantly better than those of other groups. The centrifugal supernatant also has the effect of promoting the formation of a crust, but the effect is slightly weaker than that of the complete bacterial liquid.

[0022] (3) The sand barrier formed by the sand-fixing method of the present application has a high dry density, significantly improving the stability and wind erosion resistance of the sand barrier. Compared with physical barriers and biological measures, the sand-fixing method provides more durable sand-fixing effects and is suitable for extreme environments, and can maintain good sand-fixing effects in arid and semi-arid areas. The present application found through desert field tests that the 75% bacterial liquid treatment group had the least wind-sand accumulation from the beginning to the end, with a sand accumulation of 127.21g in 40d, which was significantly lower than that of the pure water treatment group and the positive control group, indicating strong sand-fixing ability. The use of 75% bacterial liquid for sand crust formation has the advantages of short crust formation time and high strength, and the method has good ecological adaptability and operability, which can provide a new technical path for desertification prevention and control and microbial sand barrier construction. BRIEF DESCRIPTION OF DRAWINGS

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 OD values ​​of TRM82006 bacterial culture at different culture times 600 Value statistics chart.

[0025] Figure 2 This is a colony morphology diagram of Paenibacillus oleatilyticus TRM82006.

[0026] Figure 3 This is a scanning electron microscope image of Paenibacillus oleatilyticus TRM82006.

[0027] Figure 4 Phylogenetic tree of Paenibacillus oleatilyticus TRM82006.

[0028] Figure 5 The images show the crust formation in the sand tanks on day 10 for different treatment groups.

[0029] Figure 6 The image shows the thickness of the sand crust on day 10 for different treatment groups.

[0030] Figure 7 The graph shows the hardness of the sand crust on day 10 for different treatment groups.

[0031] Figure 8 This is a scanning electron microscope image of sandy soil crust. Figure 8 In the image, A represents a scanning electron microscope image of uncrusted sand. Figure 8 B in the image is a scanning electron microscope image of the crusted sand.

[0032] Figure 9 This is a graph showing the statistical results of wind erosion sand volume at different monitoring times during a desert field experiment. Detailed Implementation

[0033] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially. Unless otherwise specified, the percentages in the following embodiments refer to mass percentages.

[0034] Each liter of Gause No. 1 medium includes soluble starch (20 g), KNO3(1 g), K2HPO4(0.5 g), MgSO4·7H2O (0.5 g), NaCl (0.5 g), FeSO4·7H2O (0.01 g), pH = 7.5.

[0035] Example 1

[0036] This example provides the screening and identification of strain TRM82006.

[0037] 1. Screening of strain TRM82006

[0038] In September 2022, soil samples were collected from the rhizosphere of Haloxylon ammodendron in the Taklimakan Desert. High-yield exopolysaccharide-producing strains were screened by LB aniline blue drawing method. Inoculation loop was used to gently touch the colonies to determine whether they had "stickiness". Any colony determined to have "stickiness" was a potential exopolysaccharide (EPS) producing strain. EPS was extracted from the fermentation broth by low-temperature alcohol precipitation separation method, and the content of exopolysaccharide secreted by the potential producing strain was determined by anthrone-sulfuric acid method to determine that strain TRM82006 was a high-yield exopolysaccharide-producing strain. The TRM82006 bacterial liquid screened was inoculated in Gause No. 1 medium, and cultured in a constant temperature shaker at 30°C and 120 rpm for 72 h. During the culture, the OD 600 value and pH value of the bacterial liquid in the culture medium were measured every two hours to monitor the growth of TRM82006. The OD 600 values of the bacterial liquid at different culture times are shown in Figure 1 . After the OD 600 value reached 1.0, the culture was terminated. If the culture medium changed from transparent to turbid and there were no foreign bacteria, it indicated that the bacteria were successfully activated, and TRM82006 seed liquid was obtained. The seed liquid was inoculated in a 100 L fermenter at a volume ratio of 1% at 30°C and 120 rpm for 48 h to obtain bacterial liquid, which was stored at 4°C for standby.

[0039] 2. Identification of strain TRM82006

[0040] (1) Morphological identification

[0041] Colony morphology: TRM82006 grew as small transparent protruding colonies in the early stage, and as round, yellow transparent protruding, gelatinous, sticky or more or less, large bacterial bodies Figure 2 ) in the later stage. Figure 3

[0042] (2) Molecular biological identification

[0043] ​The 16S rRNA of the strain TRM82006 was sequenced. First, the total DNA was extracted by CBTA method, and then specific amplification was carried out by PCR using primers. The PCR reaction system: 2x EasyTaq SuperMix 12.5 μL, 27F 0.5 μL, 1492R 0.5 μL, DNA 50 ng, sterile water to 25 μL. The PCR reaction conditions: 94℃ 5 min, 94℃ 30 s, 54℃ 30 s, 72℃ 1 min 30 s, 30 cycles; 72℃ 10 min, 4℃ storage.

[0044] Upstream primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0045] Downstream primer 1492R: 5'-GGTTACCTTGTTACGACTT-3'.

[0046] The amplification product was tested on a 1% agarose gel, and the bright band amplification product was sent to Shanghai Qikexin Biotechnology Co., Ltd. for sequencing. The sequencing result showed that the 16S rRNA sequence of the strain TRM82006 was as shown in SEQ ID NO: 1. The sequencing result was compared in the EZBioCloud database, and it was found that the strain TRM82006 had a similarity of 98.37% with the known strain (Paenibacillus oleatilyticus SM69), and the phylogenetic tree was constructed as shown in Figure 4 , which indicated that the strain TRM82006 was a Paenibacillus strain.

[0047] Example 2

[0048] This example provides the influence of Paenibacillus oleatilyticus TRM82006 on soil crust formation.

[0049] Paenibacillus oleatilyticus TRM82006 was inoculated in sterilized Gao's No. 1 medium at an inoculation amount of 2%, and cultured at 30℃ with shaking at 120 r / min for 72 h to obtain a bacterial liquid with an OD 600 value of 2.0.

[0050] The sand tank test was carried out in a controllable indoor environment. The test involved 13 treatment groups, including four bacterial liquid groups, four supernatant groups, and five control groups. The bacterial liquid groups were diluted to 25%, 50%, 75%, and 100% (v / v), respectively. The supernatant groups were obtained by centrifuging the bacterial liquid at 12,000 r / min for 30 min, and then diluted to 25%, 50%, 75%, and 100% (v / v), respectively. The control groups included a sterilized Gao's No. 1 medium group (sterilized Gao's No. 1 medium was diluted to 25%, 50%, 75%, and 100%, respectively) and a deionized water group. Each group had three replicates. The sand tank used in the test was filled with wind-blown sand of the same volume (half the height of the tank), and 20 mL of the corresponding treatment liquid was applied to each tank. The spray device was used to uniformly spray the sand surface. The test was incubated in an artificial climate chamber with a temperature of 25-28°C and a humidity of 60-70%. Crust observation and sampling analysis were performed every 10 days. The crust thickness (measured using an electronic digital caliper at five points along the diameter) and hardness (recorded by vertically piercing with an electronic scale + 0.5 mm metal nib) were measured. The observation results are shown in Figure 5 , the measurement results are shown in Figure 6 and Figure 7 , and the scanning electron microscope observation of the crust and uncured sand is shown in Figure 8 .

[0051] As shown in Figure 6 and Figure 7 , the bacterial liquid treatment group can form continuous biological crust within 10 days. The average thickness and pressure-bearing capacity (hardness) of the crust in the 75% bacterial liquid treatment group are significantly better than those of the other groups. The centrifuged supernatant also has a promoting effect on crust formation, but the effect is slightly weaker than that of the complete bacterial liquid. The sterilized Gao's No. 1 medium contains more starch and also has a promoting effect on crust formation, but the stability of the crust is weakened compared with the experimental groups, and the deionized water group does not form a crust. Therefore, Paenibacillus oleatilyticus TRM82006 is suitable for application as a desert microbial sand fixation material.

[0052] Example 3

[0053] This example provides a desert field test of the influence of Paenibacillus oleatilyticus TRM82006 on soil crust formation.

[0054] The bacterial liquid was prepared in the same way as in Example 2, and was diluted to 25%, 50%, and 75%, respectively. A pure water treatment group was set as a control group, and a positive control group was planted with Haloxylon ammodendron trees. Each treatment had three replicates.

[0055] The Taklimakan Desert hinterland sandy land is selected as the test site, and an aeolian erosion monitoring area is set up. The aeolian erosion monitoring area is set up with 5 sample plots (5m x 5m), and a creeping sand collecting barrel is arranged at the center. In order to prevent the moving sand from burying, a windbreak net barrier is set up around the sample plot. A manual sprayer is used to spray each treatment liquid on the bare sand surface. The aeolian erosion monitoring area is monitored every 10 days, and the sand collecting samples are collected for 70 days, a total of 7 times of sampling. The accumulated sand weight (unit: g) in the sand collecting barrel is recorded for each sampling of each group to reflect the change of aeolian erosion sand amount, and the statistical results are shown in Table 1. 2 As shown in Table 1. Figure 9 As shown in Table 1.

[0056] As shown in Table 1. Figure 9 It can be seen that the pure water treatment group appears a sand collecting peak of 397.48g and 459.80g at 20d and 40d respectively, indicating that the wind erosion is the most serious under the condition of no treatment; the positive control group has an accumulated sand amount between the pure water group and the bacterial liquid treatment group, indicating that planting haloxylon trees has a certain sand control effect; the 25% bacterial liquid treatment group has a slightly decreased sand collecting amount compared with the pure water treatment group, but still reaches 182.08g at 40d, and has limited sand control ability; the 50% bacterial liquid treatment group rapidly decreases after reaching a peak of 303.02g at 40d, showing a certain sand control effect; the 75% bacterial liquid treatment group has the least wind-sand accumulation from the beginning to the end, and the sand collecting amount at 40d is 127.21g, which is significantly decreased compared with the sand collecting amount of the pure water treatment group and the positive control group, and shows the strongest sand fixation ability.

[0057] The above description describes the basic principles, main features and advantages of the present application. The above examples and descriptions are only used to describe the preferred embodiments of the present application, and the present application is not limited by the above examples. Without departing from the spirit and scope of the present application, various changes and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the scope of protection of the present application.

Claims

1. A strain that produces extracellular polysaccharides, characterized in that, The strain described is Paenibacillus taklamakanensis TRM82006, deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No. 66363.

2. The strain according to claim 1, characterized in that, The nucleotide sequence of the 16S rRNA of the strain is shown in SEQ ID No:

1.

3. The application of at least one of the strain of claim 1, the fermentation broth of the strain, and the metabolite of the strain in sand fixation.

4. The application according to claim 3, characterized in that, After use, it promotes the crusting of sandy soil.

5. The application according to claim 3, characterized in that, After use, the movement of sand particles is reduced, and wind erosion is decreased.

6. The application according to claim 3, characterized in that, The preparation method of the fermentation broth includes: inoculating Paenibacillus taklamakanensis TRM82006 at a 2% inoculation rate into Gao's No. 1 medium and culturing it in a shaker at 28-30℃ and 120r / min for 72h.

7. A microbial inoculant, characterized in that, The active ingredient comprises at least one of the strain of claim 1, the fermentation broth of the strain, and the metabolites of the strain.

8. A microbial sand fixation method, characterized in that, Use the microbial agent as described in claim 6.

9. The microbial sand fixation method according to claim 8, characterized in that, The microbial agent can be sprayed directly or diluted to 25-75% before spraying.

10. The microbial sand fixation method according to claim 9, characterized in that, The spraying rate is not less than 1L / m 2 .

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