Fertilizing microorganism screening and synthetic community construction method for saline-alkali soil remediation

Abstract

The invention belongs to the technical field of microbial communities, and relates to a fertility-increasing microorganism screening and synthetic community construction method for saline-alkali soil remediation. The construction method comprises the following steps: carrying out domestication and enrichment culture on a pretreated soil sample to be repaired by using an improved culture medium to obtain a functional flora, separating to obtain a plurality of functional strains, monitoring a growth curve of the functional strains in a simulated soil solution, constructing a growth rate evaluation model, and dividing the strains into a rapid growth type strain and a slow growth type strain; the method comprises the following steps: screening strains with saline-alkaline resistance, adsorption colonization and phosphorus-solubilizing and nitrogen-fixing properties to obtain dominant target strains, and combining the target strains to obtain a synthetic microbial community containing more than one rapid or slow growth type phosphorus-solubilizing / nitrogen-fixing strain. According to the invention, the slow growth type strain is incorporated into the design of the synthetic inoculant, and forms an ecological alliance with complementary functions and growth strategies with the rapid growth type strain, and the efficient and lasting soil fertility increasing effect is achieved. The method has a wide application prospect in the fields of ecological system restoration and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial community synthesis, and particularly relates to a method for screening and constructing a synthetic community of fertilizing microorganisms for saline-alkali soil remediation. BACKGROUND

[0002] Microbial inoculants contain a variety of beneficial microorganisms (such as bacteria, fungi, etc.), which can improve soil fertility and promote crop growth through mechanisms such as nitrogen fixation, phosphorus dissolution, and growth promotion, and have important significance for reducing fertilizer application and improving soil ecological environment. For example, nitrogen-fixing bacteria can convert atmospheric nitrogen into a form available to plants, while phosphorus-dissolving bacteria can activate insoluble phosphorus in the soil and enhance the availability of phosphorus nutrients. Although the theoretical advantages of microbial inoculants are significant, their field application effects often face the bottleneck of insufficient stability and sustainability. This is mainly because the existing inoculants have the following shortcomings in screening and construction strategies: Traditional screening methods rely heavily on high selective pressure media (such as nitrogen-free medium for screening nitrogen-fixing bacteria and PKO medium for screening phosphorus-dissolving bacteria). This screening paradigm, which focuses on "growth rate" as the core indicator, systematically favors strains that rapidly proliferate in homogeneous and nutrient-rich conditions. However, such strains often allocate most of their energy to growth and reproduction, leaving deficiencies in environmental tolerance, substrate utilization breadth, stress-resistant substance synthesis, and community interaction. More importantly, a large number of slow-growing strains with important ecological functions (such as synthesis of extracellular polymers, antibiotics, and signaling molecules) are overlooked, leading to the inability of the selected strains to stably colonize and construct interaction networks in complex field environments, resulting in the phenomenon of "laboratory top students, field failures."

[0003] At the same time, existing inoculants often use single-function bacteria or fast-growing strains selected in the laboratory for mechanical combination, lacking systematic consideration of niche complementarity and interspecific interaction between strains. In actual application, these strains with highly overlapping niches will be involved in fierce competition for nutrients and space, leading to functional inhibition and even community collapse, and cannot form stable and synergistic functional associations. Although synthetic biology advocates the design of artificial synthetic communities based on functional complementarity, redundancy, and environmental adaptability, the complex and unpredictable interactions between microbial species (such as competition, symbiosis, and antagonism), and the highly complex metabolic network regulation make rational design difficult, often resulting in uncontrolled communities or functional disorders. SUMMARY

[0004] The purpose of the present application is to provide a method for screening and constructing a synthetic community of fertilizing microorganisms for saline-alkali soil remediation, thereby overcoming the shortcomings of the prior art, incorporating slow-growing functional bacteria into synthetic inoculant design, screening high-performance fast and slow-growing functional strains, and constructing a synthetic community, which has the advantages of long-term stability, environmental adaptability, and functional maximization.

[0005] To achieve the above object, the technical scheme of the present application is: In a first aspect, the present application provides a method for constructing a synthetic microbial community, comprising the following steps: The pretreated soil sample to be repaired is subjected to enrichment domestication culture using a selective medium, a plurality of functional strains are isolated and obtained, and the growth curves of the strains in simulated soil solution are monitored; Based on the growth curves, a growth rate evaluation model is constructed, and the strains are divided into fast-growing strains and slow-growing strains; The fast-growing strains and slow-growing strains are screened in terms of salt-tolerant, adsorption, planting, and phosphorus-dissolving and nitrogen-fixing performance to obtain target strains; The target strains are combined to obtain a synthetic microbial community. The synthetic microbial community contains more than one fast-growing strain and more than one slow-growing strain.

[0006] The inventors found in the research process that slow-growing strains often have more stable metabolic product expression and long-term planting ability, can maintain basic function output during environmental fluctuation period, and improve the long-term stability and repair persistence of the community. Secondly, such strains usually have a wider niche distribution width and better adaptability to environmental factors and microbial communities, can maintain homeostasis under limited resources or stress conditions, thereby buffering the functional fluctuations of fast-growing strains under high competitive pressure. Thirdly, the synergistic effect of slow-growing strains can enhance the metabolic coupling and mutualistic relationship of the community, promote nutrient cycling and slow release and utilization of key elements such as nitrogen and phosphorus, thereby improving the synergistic effect of the overall functional spectrum and enhancing the comprehensive effect of soil repair. Finally, the combination of the related functional strategies of fast and slow-growing strains can achieve the dual goals of rapid establishment of functional output in the early stage and maintenance of stable functional expression in the later stage, improve the controllability, predictability, and feasibility of engineering amplification of the repair process.

[0007] Although existing literature generally recognizes that fast-growing strains have a higher intrinsic growth rate, the evaluation index is too single, and the numerical value is only a relative concept, lacking clear boundaries. The Gr evaluation model integrates multiple key parameters related to growth, overcoming the one-sidedness of single trait indicators, and can more comprehensively reflect the multi-dimensional characteristics of microbial reproductive capacity. This scoring system quantifies multi-dimensional growth characteristics into standardized numerical values that can be directly compared, providing a unified and intuitive evaluation standard for the reproductive capacity of different strains, significantly improving the scientificity and practicality of the evaluation results. When synthesizing, more energy should be allocated to slow-growing strains involved in community interaction.

[0008] In some other embodiments, the pretreatment method of the soil sample to be remediated is removing sundries, naturally air-drying, sieving and mixing; the pH of the soil to be remediated is 7-9.5, and the concentration of soluble salt is 2-6 g / kg.

[0009] Specifically, the pH of the soil to be remediated is 8.68±0.06, and the concentration of soluble salt is 3.12±0.15 g / kg. Meanwhile, the concentration of organic matter in the soil to be remediated is 3.2±0.19 g / kg, the concentration of total phosphorus is 0.22±0.04 g / kg, the concentration of total nitrogen is 0.18±0.03 g / kg, the concentration of total potassium is 3.68±0.23 g / kg, the concentration of available phosphorus is 1.46±0.15 mg / kg, the concentration of available nitrogen is 15.63±0.62 mg / kg, and the concentration of available potassium is 20.25±0.35 mg / kg.

[0010] In some other embodiments, the domestication medium is PVK, Ashby solid medium or improved formula based thereon, or nitrogen-deficient / nitrogen-lean / increased insoluble phosphorus simulated soil, in which domestication and enrichment of slow-growing bacteria are carried out; the selective medium for isolation of functional bacteria is PVK, Ashby solid medium. The pH of the simulated soil solution for performance verification of isolated functional bacteria is 7-9.5, and the salinity is 0.2%-0.5%. The commonly used LB solution is replaced by the simulated soil solution, which is closer to the real soil sample to be remediated, thereby improving the accuracy of screening.

[0011] In some other embodiments, the growth rate evaluation model Gr is based on the highest OD 600 , intrinsic growth rate, lag phase time, time to reach stationary phase, 1 / 2 maximum growth time, and log mid-phase time, which are growth parameters, and is constructed by assigning specific weights to each parameter.

[0012] The calculation formula of Gr is as follows: Gr=OD Max × 0.46+μ×0.45-t1×0.075-t2×0.005-t3×0.005-t4×0.005; Wherein, Gr is the growth rate evaluation model of the strain, μ is the intrinsic growth rate, t1 is the lag phase time, t2 is the time to reach the stationary phase, t3 is the 1 / 2 maximum growth time, t4 is the time to reach the log mid-phase time, OD Max is the highest OD 600 .

[0013] The present application ensures that the strain is not only powerful in the soil environment to be repaired, but also has excellent environmental adaptability and stable colonization in subsequent application, thereby fundamentally ensuring its efficiency in practical application. The multi-stage evaluation method (salt-tolerant ability, adsorption colonization ability and phosphorus-dissolving and nitrogen-fixing performance) can efficiently eliminate strains that are only outstanding in one aspect but lack comprehensive ability, accurately lock target strains that are more likely to survive and have more durable functions in complex environments, and greatly improve the success rate and predictability of synthetic community construction.

[0014] In some other embodiments, the fast-growing strain is a strain with 0.5 < Gr < 1, and the slow-growing strain is a strain with 0 < Gr < 0.5.

[0015] In some other embodiments, the slow-growing strain is Serratia odorifera (S. odorifera) with a strain number of ATCC 33077. Serratia odorifera Bacillus megaterium ) 、 Bacillus megaterium (B. megaterium) with a strain number of ATCC 14581 Stenotrophomonas maltophilia ) 、 Stenotrophomonas maltophilia (S. maltophilia) with a strain number of ATCC 13652 Serratia rhizosphaerae ) 、 Serratia rhizophila (S. rhizophila) with a strain number of CIP 105886 Bacillus subtilis ) Sinorhizobium meliloti Bacillus subtilis (B. subtilis) with a strain number of CIP 52.65 Atlantibacter hermanni ) Paenibacillus polymyxa Rhizobium meliloti (R. meliloti) with a strain number of CIP 105263

[0016] In some other embodiments, the fast-growing strain is Hermannella atlantica (H. atlantica) with a strain number of CIP 105.883 Bacillus haikouensis Stenotrophomonas hibiscicola ) 、 Paenibacillus polymyxa (P. polymyxa) with a strain number of CIP 105.883 Halomonas axialensis ) 、 Bacillus haikouensis (B. haikouensis) with a strain number of CIP 105.883 Klebsiella variicola Serratia odorifera ) 、 Stenotrophomonas rhodocacia (S. rhodocacia) with a strain number of CIP 105.883 Bacillus megaterium ) Stenotrophomonas maltophilia Halomonas axeniana (H. axeniana) with a strain number of CIP 105.883 Serratia rhizosphaerae ) and Klebsiella variicola (K. variicola) with a strain number of CIP 105.883

[0017] In the second aspect, the present application provides a microbial combination for saline-alkali soil remediation, comprising a fast-growing strain and a slow-growing strain. The slow-growing strain is selected from Serratia odorifera (S. odorifera) with a strain number of ATCC 33077 Bacillus subtilis ) The fast-growing strain is Bacillus megaterium (B. megaterium) with a strain number of ATCC 14581 、 ) The slow-growing strain is Serratia odorifera (S. odorifera) with a strain number of ATCC 33077 Sinorhizobium meliloti ) The fast-growing strain is Bacillus megaterium (B. megaterium) with a strain number of ATCC 14581 、 The slow-growing strain is Serratia odorifera (S. odorifera) with a strain number of ATCC 33077 Atlantibacter hermanni) whose strain number is CGMCC 1.1788 、 Rhizosphere Serratia marcescens Paenibacillus polymyxa ) whose strain number is CGMCC 1.18473, Bacillus subtilis Bacillus haikouensis ) whose strain number is CCTCC AB 2021503, Sinorhizobium meliloti Stenotrophomonas hibiscicola ) whose strain number is CCTCC AB 205596; The fast-growing strain is selected from the group consisting of Hermannella atlantica Halomonas axialensis ) whose strain number is ATCC 33650 、 Paenibacillus polymyxa Klebsiella variicola ) whose strain number is ATCC 842 、 Bacillus haikouensis Serratia odorifera ) whose strain number is CCTCC AB 2014076 、 Xenorhabdus rhodii Atlantibacter hermanni ) whose strain number is ATCC 19867, Halomonas axinifodinis Paenibacillus polymyxa ) whose strain number is CCTCC AB 2020019, and Klebsiella variicola Bacillus haikouensis Bacillus megaterium ) whose strain number is CCTCC AB 2018100.

[0018] The present application realizes time-space function complementation and metabolic synergistic effect by the technical leap from simple compounding of strains to construction of ecological community. The synthetic microbial community constructed based on the ecological principles of functional complementation, functional redundancy, environmental adaptability, outstanding performance, and long-term stability forms a stable symbiotic "micro-ecological alliance" through positive interaction. The fast-growing strain rapidly establishes an ecological niche and starts the target function in the resource-rich stage due to its rapid proliferation characteristics, while the slow-growing strain maintains the system for long-term operation through high substrate affinity and stable metabolic network, forming an efficient functional relay mechanism. The community can persist in the soil for a long time and continuously exert the effect of improving soil fertility, effectively overcoming the technical bottleneck of single function and short duration of traditional microbial agents, and providing core technical support for realizing green and sustainable yield increase in agriculture.

[0019] In some other embodiments, the complex ratio of the fast-growing strain and the slow-growing strain is 1:1-1:3 to increase the long-term stability function of the slow-growing strain.

[0020] Specifically, the microbial combination for saline-alkali soil remediation comprises Serratia odorifera Serratia rhizosphaerae ) whose strain number is ATCC 33077, Hermannella atlantica Stenotrophomonas maltophiliaIts strain number is ATCC 33650, Bacillus polymyxa ( Stenotrophomonas hibiscicola Its strain number is ATCC 842 and Bacillus haikouensis ( Figure 1 Its strain number is CCTCC AB2014076, Bacillus megaterium ( Figure 2 Figure 3 Its strain number is ATCC 14581, *Serratia rhizospherea* (… Figure 4 Its strain number is CGMCC 1.18473, Stenotrophomonas maltophilia ( Figure 5 The strain numbered CGMCC 1.1788 and *Stenotrophomonas hibiscus* ( Figure 1 The strains were numbered ATCC 19867 and were mixed in the same ratio of 1:1.

[0021] Thirdly, the present invention provides the application of the microbial assemblies for saline-alkali land remediation described in the second aspect in saline-alkali land remediation.

[0022] The beneficial effects of this invention are: (1) The slow-growing strains selected by the method of this invention are characterized by robust metabolism, strong environmental adaptability, and a wide ecological niche. They can maintain basic functional output under resource fluctuations or environmental stress, effectively buffering the functional instability of fast-growing strains caused by competition or stress. The two types of strains form a functional relay mechanism of "rapid establishment - long-term maintenance" in time, ensuring the durability and reliability of the overall community performance. At the same time, slow-growing strains usually have more complex substrate utilization capabilities and metabolic complementarity, which can form an efficient metabolic coupling network with fast-growing strains. This network promotes the cycling and slow release of key elements such as carbon, nitrogen, and phosphorus, improves nutrient utilization efficiency, and thus exhibits stronger comprehensive functions in soil remediation and fertility enhancement.

[0023] (2) This invention employs a multi-dimensional evaluation system that simulates a real environment, enabling the elimination of strains that are "excellent in the laboratory but fail in the field" during the screening stage, and accurately identifying target strains with high adaptability and colonization capacity. Combining ecological principles such as functional complementarity, redundant design, and environmental adaptation, it significantly improves the success rate, controllability, and engineering scale-up potential of synthetic community construction. Furthermore, the synthetic microbial communities constructed in this invention are not only suitable for improving the quality and yield of agricultural soil, but can also be widely applied in fields such as ecosystem restoration, biomanufacturing, and pollutant degradation, demonstrating good versatility and promotional value.

[0024] In summary, the construction strategy of this invention, which moves from "strain superposition" to "system ecology," has enabled the microbial community to leap from short-term high efficiency to long-term stability, providing a reliable path for the precise application of microbial technology in the field of sustainable development. Attached Figure Description

[0025] The accompanying drawings, which form a part of this specification, are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification. The embodiments of the application, together with its

[0026] Figure 2 The method for screening and synthesizing the microbial fertilizer for saline-alkali soil remediation in embodiment 1 of the application; Figure 2 The growth curves of the 14 strains screened in embodiment 1 of the application in LB culture solution and soil simulation solution; Atlantibacter hermannii The relative abundance (colonization ability) of the 14 strains screened in embodiment 1 of the application in real soil; Figure 2 The content of water-soluble phosphorus of the 14 strains screened in embodiment 1 of the application in LB and soil simulation solution changes with time; Agrobacterium pusense The actual application effect of different grouping microbial agents in embodiment 1 of the application. DETAILED DESCRIPTION

[0027] Those skilled in the art will appreciate that the following examples are included for illustrative purposes only and should not be viewed as limiting on the scope of the application. Unless otherwise indicated, conventional conditions have been used, or those as recommended by the manufacturer have been used. Where the manufacturer of reagent is not indicated, it will be appreciated by those skilled in the art that the reagent is a conventional product available commercially.

[0028] As mentioned above, the traditional screening of nitrogen-fixing and phosphorus-dissolving bacteria generally uses nitrogen-free medium or phosphorus-dissolving circle method, which is easier to obtain functional microorganisms with high growth rate. In the subsequent evaluation process of the advantage functional microorganisms, in addition to high performance such as nitrogen fixation and phosphorus dissolution, fast growth is usually taken as an important screening index to synthesize microbial agents for soil fertilization. However, slow-growing strains are usually eliminated in such growth rate-oriented screening process because of their slow growth in the screening culture process.

[0029] To solve the above-mentioned defects, the present application proposes to incorporate slow-growing functional bacteria into the design of synthetic bacterial agents, screen high-performance fast and slow-growing functional strains, and construct synthetic communities, so that they have the advantages of long-term stability, environmental adaptation, and functional maximization. This is because slow-growing bacteria can direct more metabolic resources to extracellular polymer secretion, signal molecule synthesis, and other inter-species cooperative behaviors. They play a key role in microbial communities, can enhance the overall ecological function of the bacterial community by maintaining population stability, promoting material circulation and information exchange, and have an important role in soil fertilization. Therefore, incorporating slow-growing functional bacteria into the design of synthetic bacterial agents, and constructing an ecological alliance with fast-growing strains that complement each other in function and growth strategy, is expected to achieve more efficient and sustainable soil fertilization. This method has wide application prospects in the fields of ecosystem restoration, biological manufacturing, soil fertilization, and agricultural yield increase.

[0030] Embodiment 1 The present embodiment proposes a method for screening and constructing a fertilizing microorganism for saline-alkali soil remediation, as shown in Figure 2 The method specifically comprises the following steps: (1) Collecting soil samples to be remediated and domesticating the bacterial community under conditions of nitrogen reduction and increased insoluble phosphorus, isolating nitrogen-fixing and phosphorus-dissolving bacteria in a selective culture medium, and then culturing the strains in different environmental gradients in a microwell plate under different culture conditions (simulated saline-alkali soil solutions with different degrees of salinity and alkalinity), and continuously monitoring their growth curves; (2) Based on the monitored growth curves, obtaining the OD Max , intrinsic growth rate (μ), lag phase time (t1), time to reach the stable phase (t2), 1 / 2Max time (t3), and time to reach the mid-log phase (t4) of each strain, and constructing a strain growth rate Gr evaluation model, and preliminarily screening and classifying the strains into fast-growing and slow-growing strains according to the calculation results of the Gr model.

[0031] Specifically, the strain growth rate Gr evaluation model is constructed as follows: Gr=OD Max × 0.46+μ×0.45-t1×0.075-t2×0.005-t3×0.005-t4×0.005.

[0032] (3) Further screening the preliminarily screened strains by constructing a niche map to determine the target strains.

[0033] Specifically, the niche map construction fully considers the ecological amplitude of the strain, the growth rate, and the adsorption capacity (colonization capacity), and the superior functional strains are screened out through multi-stage screening. In the strain function evaluation, the real environment situation is mainly considered. The purpose is to break through the evaluation barrier between the laboratory and the field application, and to make the function verification more focused on the actual effectiveness of the strain in specific applications such as soil remediation, agricultural yield increase, or pollutant degradation.

[0034] According to the principle of microbial community construction, the present application scientifically combines the slow and fast growth functional bacteria of different growth rates, and monitors the change rule of the key fertility index in the soil with time, so as to master the advantage of slow growth type functional bacteria in soil fertilization. The slow growth type functional bacteria is incorporated into the synthetic community system, and the synthetic microbial agent is prepared by coupling the fast and slow growth type microorganisms, so as to realize the purpose of soil fertilization.

[0035] In order to more clearly illustrate the embodiments, the design and construction method of the microbial synthetic community is described as follows based on the actual situation and the growth kinetics of the strain: (1) The soil sample to be repaired was collected from a moderate saline-alkali land in Dongying, Shandong Province (37°48'13''N; 118°36'44''E), and the physicochemical properties such as pH, soil soluble salt, soil organic matter, total phosphorus, total nitrogen, total potassium, available phosphorus, available nitrogen, and available potassium were determined. The test results of the related physicochemical properties of the soil are shown in Table 1. Among them, the determination method of soil pH refers to NY / T 1121.2-2006, the determination method of soil soluble salt (salt content) refers to LY / T 1251-1999, the determination method of soil total phosphorus refers to NY / T 88-1988, the determination method of soil total nitrogen refers to LY / T 1228-2015, the determination method of soil organic matter refers to NY / T 85-1988, the determination method of soil total potassium refers to GB 9836-88, the determination method of soil available phosphorus refers to NY / T 1121.7-2014, the determination method of soil available nitrogen (total nitrogen) refers to LY / T 1228-2015, and the determination method of soil available potassium refers to NY / T-889-2004. The soil moisture content is determined by oven drying, and the soil porosity is indirectly determined by soil particle density. After removing plant roots, stones and other impurities from the collected soil, the pretreated soil sample is dried under natural conditions, mixed uniformly through a 2 mm screen, and prepared for use.

[0036] Table 1 Related physicochemical properties of the soil to be repaired

[0037] (2) Take 10 g of pretreated and acclimated soil sample, add 90 mL of sterile deionized water, and shake vigorously on a shaker for 20-30 min to fully disperse the microorganisms. Then dilute with sterile water in turn to prepare a concentration gradient of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 . Take 10 -2 to 10 -7 of the dilutions 100 μL and inoculate into PVK solid medium, evenly spread with a spreader, and incubate at 30°C for 3-5 d to isolate functional bacteria. Then, select single colonies and purify on PVK solid medium for 4 times or more to finally obtain 14 strains of phosphorus-solubilizing bacteria. Use a variety of different culture conditions, i.e. LB solution and simulated soil solution, and set different high and low salt gradients in each of the two solutions, i.e. pH 7.0, salinity 0.2%; pH 8.5, salinity 0.3%; pH 9.5, salinity 0.5%, culture the strains, and real-time detect the growth curves of each strain on a 96 plate (as shown in Pseudarthrobacter oxydans ). Among them, the LB culture solution can be commercialized, and the composition of the soil simulation solution is shown in Table 2.

[0038] Table 2 Composition of simulated soil solution

[0039] (3) According to the highest OD 600 , intrinsic growth rate (μ), lag phase time (t1), time to reach the stationary phase (t2), 1 / 2max time (t3), and logarithmic mid-period time (t4) of each strain, assign weights and calculate the growth rate index Gr, wherein the calculation formula of Gr is as follows: Gr = OD Max × 0.46 + μ × 0.45 - t1 × 0.075 - t2 × 0.005 - t3 × 0.005 - t4 × 0.005.

[0040] The Gr evaluation model of the strain growth rate is obtained by the inventors according to the growth curves of 14 strains of phosphate-solubilizing bacteria obtained by screening in moderate saline-alkali soil in different saline-alkali environments (mild: pH: 7-8.5, salinity 1-3 g / kg; moderate: pH: 8.5-9.5, salinity 3-5 g / kg; severe: pH: > 9.5, salinity > 5 g / kg), and the original load of PC1 (the first principal component in a set of linearly independent new variables (i.e. principal components) obtained by linear transformation of the original data) is normalized as the weight of each parameter. The calculation results show that the Gr value > 0.5 represents a fast-growing strain, and vice versa, thereby classifying the strains into fast-growing and slow-growing types.

[0041] According to Figure 2 and combined with the Gr index calculation, the strains are classified into two categories, specifically, the fast-growing strains are Paenibacillus polymyxa ( Figure 2 Strain 2 in Table 1, Bacillus haikouensis ( Figure 2 Strain 4 in Table 1, Limimaricola soesokkakensis ( Figure 2 Strain 5 in Table 1 Stenotrophomonas hibiscicola ( Figure 2 Strain 6 in Table 1, Serratia odorifera ( Figure 2 Strain 7 in Table 1, Bacillus megaterium ( Figure 2 Strain 12 in Table 1, Serratia rhizosphaerae ( Figure 2 Strain 14 in Table 1; and the slow-growing strains are Micrococcus flavus Figure 2 ( Mesobacillus jeotgali Strain 1 in Table 1, Figure 2 ( ​ Strain 3 in Table 1, ​ ​ ( ​ Strain 8 in Table 1, ​ ( ​ Strain 9 in Table 1, ​ ​ ( ​ Strain 10 in Table 1 Stenotrophomonas maltophilia ( Figure 2 Strain 11 in Table 1, Bacillus pumilus ( Figure 2 Strain 13 in Table 1.

[0042] (3) Evaluation of strain adsorption performance and colonization effect on soil particles: after measuring the growth curve of the strain, the 96-well plate is emptied and sterile water is added, and the OD 600The amount of bacteria eluted from the surface of the well plate was evaluated by measuring the OD value of the eluate. We observed that the elution efficiency of each strain was different, and the strains with lower elution amounts may have a stronger tendency to adhere to the surface.

[0043] After the strains were added to the soil, the colonization of the strains on the soil particles was evaluated. The specific process was as follows: 250 g of moderately saline-alkali soil (pH 8.7, salinity 3.1 g / kg) to be treated was placed in a disposable flowerpot. Each target strain was inoculated in a liquid medium and cultured to the logarithmic growth phase. The bacterial cells were collected by centrifugation and prepared into an OD 600 of about 0.8. All 8 candidate strains were mixed in equal volumes, and 25 μL of the bacterial suspension was inoculated into each g of soil. The soil was watered with pure water every 1-2 days, and the soil was taken for 16S RNA detection on days 3, 5, 8, 12, 18, 27, and 30. The colonization ability of the strains in the soil was determined by quantifying the strains over time, as shown in Figure 3 .

[0044] (4) Evaluation of the strain's own phosphorus solubilizing ability: each single strain was placed in LB and simulated soil solution (the composition is shown in Table 2) for amplification, and then calcium phosphate insoluble powder was added. The phosphorus content of the bacterial solution was measured after being filtered with a 0.22 μm filter at 1, 3, 6, 9, 12, and 15 days, and the OD600 value of the strain was also detected (excluding the effect of insoluble phosphorus), to determine the strength of the strain's own phosphorus solubilizing ability, as shown in Figure 4 .

[0045] (5) Evaluation of the strain's ability to cooperate with other strains: each single strain was mixed with the original soil bacteria at a ratio of 1:1, and the phosphorus content of the bacterial solution was measured after being filtered with a 0.22 μm filter at 1, 3, 6, 9, 12, and 15 days, to determine the strain's potential ability to cooperate with other strains, as shown in Table 3.

[0046] Table 3 Cooperation ability of single strains with original soil bacteria

[0047] The experimental results shown in Figure 4 indicate that the strains Bacillus haikouensis, Serratia rhizosphaerae all reached a relatively high OD 600The OD600 value showed good growth ability; however, in simulated soil solution, the OD600 value dropped to around 0.35. This significant difference in growth indicates that the LB component differs too much from the actual environment and cannot accurately reflect the actual survival and growth ability of the strain in real soil environment. Therefore, this embodiment verifies that using simulated soil solution to approximate real environmental conditions for strain screening and efficacy evaluation is crucial for predicting and ensuring its effectiveness in practical applications.

[0048] Eight common phosphate-solubilizing bacteria were identified using Gr index calculation and functional identification. Among them, the rapid-growing bacteria were: Atlantibacter hermanni, Paenibacillus polymyxa, Bacillus haikouensis, Stenotrophomonas hibiscicola Slow-growing bacteria are Serratia odorifera, Bacillus megaterium, Stenotrophomonas maltophilia, Serratia rhizosphaerae.

[0049] (6) After steps (3) to (5), these 8 phosphate-solubilizing strains were found to have advantages such as high salt and alkali tolerance, strong function, strong adsorption and colonization ability, and obvious growth rate differentiation, which are in line with the construction principles of synthetic communities (i.e. functional complementarity, functional redundancy, environmental adaptability, outstanding performance, and long-term stability).

[0050] (7) Take 250 g of moderately saline-alkali soil (pH: 8.7, salinity 3.1 g / kg) to be treated and place it in a disposable flowerpot. Inoculate each target strain into liquid culture medium and culture until the logarithmic growth phase. Collect the bacterial cells by centrifugation and prepare the optical density (OD) value with sterile water. 600 The working bacterial solution is uniformly about 0.8.

[0051] Strains of the same genus were purchased from the strain repository for subsequent experimental verification. The purchased strains include: *Serratia odorata* (…). Serratia odorifera Its strain number is ATCC 33077, *Atlanticella Hermannii* (…). Atlantibacter hermanni Its strain number is ATCC 33650, Bacillus polymyxa ( Paenibacillus polymyxa Its strain number is ATCC 842 and Bacillus haikouensis ( Bacillus haikouensis Its strain number is CCTCC AB2014076. Bacillus megaterium ( Bacillus megaterium Its strain number is ATCC 14581, *Serratia rhizospherea* (… Serratia rhizosphaerae Its strain number is CGMCC 1.18473, Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia The strain numbered CGMCC 1.1788 and *Stenotrophomonas hibiscus* ( Stenotrophomonas hibiscicola One or more of the strains are numbered ATCC 19867.

[0052] Selecting from the alternative strain library: high Gr group: strains with high growth rate and high biomass in simulated soil solution Atlantibacter hermanni, Paenibacillus polymyxa, Bacillus haikouensis, Stenotrophomonas hibiscicola low Gr group: strains with low growth rate and low biomass in simulated soil solution Serratia odorifera, Bacillus megaterium, Stenotrophomonas maltophilia, Serratia rhizosphaerae mixed group: 1:1 mixture of all 8 alternative strains. The working bacterial solution of each strain in the above groups was mixed in equal volume ratio to prepare a composite bacterial suspension with the same concentration. Then, the composite bacterial suspension of each group was inoculated into the prepared soil at the same inoculation amount (25 μL of bacterial suspension per g of soil). Soil without inoculation was set as a blank control. The soil was watered with pure water every 1-2 days, and 2.5 g of soil was taken on days 3, 5, 8, 12, 18, 27, and 40 for detection of available phosphorus content to evaluate the actual application effect of different groups of bacterial agents.

[0053] It can be seen from Figure 5 that under the condition of consistent inoculation amount, there is a significant difference in the available phosphorus content of the soil in different treatment groups. Specifically, by day 18, the available phosphorus content of the mixed group reached 5.3 mg / kg, while that of the high Gr group was about 2.5 mg / kg, and that of the low Gr group was about 3.1 mg / kg; by day 40, the available phosphorus content of the mixed group further increased to about 5.8 mg / kg, that of the high Gr group was about 3.7 mg / kg, and that of the low Gr group was about 4.2 mg / kg. The experiment showed that the combination of slow-growing strains as bacterial agents had better fertilization effect than the combination of fast-growing strains, and the mixed group coupled the synergistic effect of fast start and persistent performance, showing the best and stable phosphorus solubilization effect in the later culture period, significantly better than the simple application of single strategy. Therefore, it is necessary to include slow-growing functional bacteria in the design of synthetic bacterial agents.

[0054] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for constructing a synthetic microbial community, characterized in that, Includes the following steps: The pretreated soil samples to be remediated were enriched using selective culture medium to isolate multiple functional strains, and the growth curves of each strain in simulated soil solution were monitored. A growth rate evaluation model was constructed based on the growth curve, and the strains were divided into fast-growing strains and slow-growing strains. The target strains were obtained by screening fast-growing and slow-growing strains based on their salt and alkali tolerance, adsorption and colonization, and phosphorus and nitrogen fixation properties. The synthetic microbial community is obtained by combining the target strains; the synthetic microbial community contains more than one fast-growing strain and more than one slow-growing strain.

2. The method for constructing synthetic microbial communities according to claim 1, characterized in that, The pretreatment method for the soil samples to be remediated is to remove impurities, air dry naturally, and sieve and mix thoroughly. The soil to be remediated has a pH of 7-9.5 and a soluble salt concentration of 2-6 g / kg.

3. The method for constructing synthetic microbial communities according to claim 1, characterized in that, The simulated soil solution has a pH of 7-9.5 and a salinity of 0.2%-0.5%.

4. The method for constructing a synthetic microbial community according to claim 1, characterized in that, The growth rate evaluation model Gr is based on the highest OD of the strain. 600 The growth parameters, such as intrinsic growth rate, lag period, time to reach stationary phase, half-maximum growth time, and logarithmic mid-term, are constructed by assigning specific weights to each parameter.

5. The method for constructing a synthetic microbial community according to claim 1, characterized in that, The fast-growing strain is a strain with a growth rate of 0.5 < Gr < 1, and the slow-growing strain is a strain with a growth rate of 0 < Gr < 0.

5.

6. The method for constructing a synthetic microbial community according to claim 5, characterized in that, The slow-growing strain is *Serratia odorata* (…). Serratia odorifera ) 、 Bacillus megaterium ( Bacillus megaterium ) 、 Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia ) 、 Rhizosphere Serratia ( Serratia rhizosphaerae Bacillus subtilis ( Bacillus subtilis ) and Alfalfa rhizobia ( Sinorhizobium meliloti) One or more of them.

7. The method for constructing a synthetic microbial community according to claim 5, characterized in that, The fast-growing strain is *Atlanticella Hermannii* (Hermannii bacillus). Atlantibacter hermanni ) 、 Polymyxin Bacillus ( Paenibacillus polymyxa ) 、 Haikou Bacillus ( Bacillus haikouensis ) 、 Hibiscus stenotrophomonas ( Stenotrophomonas hibiscicola ), Haloxylon ammodendron ( Halomonas axialensis ) and Klebsiella variegata ( Klebsiella variicola One or more of the following.

8. A microbial assemblages for saline-alkali land remediation, characterized in that, Including fast-growing strains and slow-growing strains; The slow-growing strain was selected from Serratia odorata (Serratia odorata). Serratia odorifera Its strain number is ATCC33077 、 Bacillus megaterium ( Bacillus megaterium Its strain number is ATCC 14581 、 Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia Its strain number is CGMCC 1.1788 、 Rhizosphere Serratia ( Serratia rhizosphaerae Its strain number is CGMCC 1.18473. Bacillus subtilis ( Bacillus subtilis Its strain number is CCTCC AB 2021503. Alfalfa rhizobia ( Sinorhizobium meliloti One or more of the strains numbered CCTCC AB 205596; The fast-growing strain was selected from *Atlanticella Hermannii* (Hermannii bacillus). Atlantibacter hermanni Its strain number is ATCC 33650 、 Polymyxin Bacillus ( Paenibacillus polymyxa Its strain number is ATCC 842 、 Haikou Bacillus ( Bacillus haikouensis Its strain number is CCTCC AB2014076 、 Hibiscus stenotrophomonas ( Stenotrophomonas hibiscicola Its strain number is ATCC 19867, *Haloxylon ammodendron*. (Halomonas axialensis) Its strain number is CCTCC AB 2020019 and Klebsiella variegata ( Klebsiella variicola) One or more of the strains are numbered CCTCC AB 2018100.

9. The microbial assemblages for saline-alkali land remediation according to claim 8, characterized in that, The ratio of the fast-growing strain to the slow-growing strain is 1:1 to 1:

3.

10. The application of the microbial ensemble for saline-alkali land remediation as described in claim 8 or 9 in saline-alkali land remediation.

Images