Acid-resistant alkali-producing bacterial combination, composite microbial inoculant and application thereof

Abstract

This invention provides an acid- and alkali-producing bacterial combination, a compound bacterial agent, and their applications, belonging to the field of microbial technology. This invention provides a bacterial combination including Bacillus subtilis with accession number CGMCC No. 29201. Bacillus sp S1 and Arthrobacterium with accession number CGMCC No. 29202 ( Arthrobacter sp S2. In this invention, both Bacillus S1 and Arthrobacter S2 are acid- and alkali-producing strains with strong environmental adaptability. Through synergistic effects via multiple pathways, including ammonia production, bicarbonate secretion, and activation of soil enzymes, the bacterial combination can significantly increase soil pH, reduce exchangeable aluminum and hydrolyzable acids, and simultaneously increase soil bicarbonate content, thus rapidly and comprehensively improving soil acidity from its root cause. This bacterial combination is of great significance for achieving green, efficient, and sustainable remediation of acidified soils.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a combination of acid- and alkali-producing bacteria, a compound bacterial agent, and their applications. Background Technology

[0002] Soil acidification is a natural or anthropogenic process in which soil loses basic ions and decreases pH due to the adsorption of hydrogen or aluminum ions. The highly reactive hydrogen and aluminum ions in acidified soil not only directly poison crop roots and inhibit their growth, but also lead to the fixation or loss of essential nutrients such as phosphorus, potassium, calcium, and magnesium in the soil, as well as an imbalance in the soil microbial community structure, suppressed enzyme activity, and a decline in ecosystem function.

[0003] Traditional methods for improving acidic soils primarily rely on the application of alkaline inorganic materials such as lime. While this method can quickly raise soil pH, it has several drawbacks: the improvement effect is not lasting and it is prone to "reversion"; excessive use may damage the soil's aggregate structure, leading to compaction; it is costly, and the long-term, large-scale mining of calcium carbonate is inconsistent with the principles of green and sustainable development. Therefore, it is imperative to develop environmentally friendly, sustainable biological improvement technologies that can comprehensively improve soil health.

[0004] Microbial remediation technology has attracted much attention due to its green, eco-compatible nature and its potential to both improve soil structure and promote crop growth. Among these, alkali-producing microorganisms can generate alkaline substances such as ammonia and bicarbonate through their metabolic activities, neutralizing soil acidity and reducing exchangeable aluminum content. Simultaneously, their life activities can secrete various enzymes and metabolites, promoting soil nutrient cycling and stimulating crop growth. Currently, some strains with alkali-producing capabilities have been reported in studies. However, existing technologies mostly focus on the screening and application of single strains, generally suffering from problems such as limited functionality, limited environmental adaptability, long remediation time, and unstable field effects. Summary of the Invention

[0005] The purpose of this invention is to provide an acid- and alkali-producing bacterial combination, a compound bacterial agent, and its application. The bacterial combination of this invention is resistant to acidic environments and can rapidly and comprehensively improve soil acidity barriers.

[0006] This invention provides a bacterial composition, including Bacillus ( Bacillus sp S1 and Arthrobacter ( Arthrobacter sp S2; the preservation number of the Bacillus S1 is CGMCC No. 29201; the preservation number of the Arthrobacter S2 is CGMCC No. 29202.

[0007] Preferably, the ratio of the effective viable number of Bacillus S1 and Arthrobacter S2 is 1:(1~2).

[0008] The present invention also provides a compound bacterial agent, the active ingredients of which include the bacterial combination described in the above scheme.

[0009] Preferably, the total viable count of the active ingredients in the compound microbial agent is not less than 1×10⁻⁶. 8 CFU / g or CFU / mL.

[0010] The present invention also provides the application of the bacterial combination or the compound microbial agent described in the above scheme in at least one of the following: 1) improving acidified soil; 2) enhancing soil fertility; 3) enhancing soil enzyme activity; 4) promoting crop growth.

[0011] Preferably, the improvement of acidified soil includes at least one of increasing soil pH, reducing soil exchangeable aluminum content, reducing soil hydrolyzable acid content, reducing total soil exchangeable acid content, and increasing soil bicarbonate content.

[0012] Preferably, the soil enzyme activity includes at least one of urease, sucrase, protease, and cellulase activities.

[0013] Preferably, the crop includes corn and / or vegetables; the promotion of crop growth includes increasing at least one of the following indicators: aboveground fresh weight, root fresh weight, plant height, and root length.

[0014] The present invention also provides a method for improving acidified soil and promoting crop growth, comprising the following steps: Apply an effective amount of the compound microbial agent described in the above scheme to the soil and / or crop rhizosphere that require improvement.

[0015] Preferably, the application method includes furrow application, hole application, drip irrigation, spraying, or seed dressing; the application rate of the compound microbial agent is 10 per acre of total live bacteria containing active ingredients. 12 Up to 10 14 CFU compound microbial agent.

[0016] This invention provides a bacterial composition, including Bacillus ( Bacillus sp S1 and Arthrobacter ( Arthrobacter spS2; the preservation number of Bacillus S1 is CGMCC No. 29201; the preservation number of Arthrobacter S2 is CGMCC No. 29202. In this invention, both Bacillus S1 and Arthrobacter S2 are acid-tolerant and alkali-producing strains with strong environmental adaptability. Through synergistic effects via multiple pathways such as ammonia production, bicarbonate secretion, and activation of soil enzymes, the bacterial combination can significantly increase soil pH, reduce exchangeable aluminum and hydrolyzable acid, and simultaneously significantly increase soil bicarbonate content, thus rapidly and comprehensively improving soil acidity from its root cause. Furthermore, this bacterial combination is a pure biological agent, free of chemical substances, and will not cause secondary pollution. Long-term application helps establish a healthy soil microbial community, achieving sustainable improvement in soil productivity, and is of great significance for the green, efficient, and sustainable remediation of acidified soils.

[0017] Biological Preservation Instructions Bacillus ( Bacillus sp S1 was deposited on December 1, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 29201.

[0018] Arthrobacter ( Arthrobacter sp S2 was deposited on December 1, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 29202. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.

[0020] Figure 1 This is a roadmap for strain screening and microbial community construction in Example 1 of the present invention; Figure 2 The images shown are SEM images of the strains screened in Example 1 of the present invention; the top three images, from left to right, represent S1 neutral, S2 neutral, and the synthetic bacterial community neutral; the bottom three images, from left to right, represent S1 acidic, S2 acidic, and the synthetic bacterial community acidic. Figure 3 In Example 2 of the present invention, the growth-promoting ability of single bacteria and synthetic bacterial groups in soil simulation experiments is compared (from left to right: CK, S1, S2, S1+S2). Detailed Implementation

[0021] This invention provides a bacterial composition, including Bacillus ( Bacillus sp S1 and Arthrobacter ( Arthrobacter sp S2; the preservation number of the Bacillus S1 is CGMCC No. 29201; the preservation number of the Arthrobacter S2 is CGMCC No. 29202.

[0022] In this invention, Bacillus S1 and Arthrobacter S2 in the bacterial assemblage were isolated from typical areas of Heilongjiang Province with severe soil acidification (long-term cultivated dryland and vegetable fields). The specific isolation and screening process was as follows: using plate screening, two functional strains that could tolerate strong acids and produce alkali were screened and isolated. After biochemical identification, they were identified as Bacillus (…). Bacillus sp S1 and Arthrobacter ( Arthrobacter sp S2; Both Bacillus S1 and Arthrobacter S2 can grow under a wide range of acidic conditions from pH 1 to 6, and have the ability to significantly increase the pH of acidic culture media.

[0023] In this invention, the nucleotide sequence of the 16S rDNA of Bacillus S1 is as shown in SEQ ID NO.1, specifically:

[0024] In this invention, the nucleotide sequence of the 16S rDNA of Arthrobacter S2 is as shown in SEQ ID NO.2, specifically:

[0025] In this invention, both Bacillus S1 and Arthrobacter S2 are acid-resistant strains with strong environmental adaptability, making them easy to scale up for fermentation production. This invention utilizes a bacterial combination composed of specific acid- and alkali-producing strains to systematically and simultaneously achieve multiple effects of "acid reduction, aluminum reduction, enzyme enhancement, and growth promotion." Through interspecies collaboration, it can more stably and efficiently address various soil obstacles. Furthermore, Bacillus S1 and Arthrobacter S2 in this invention exhibit synergistic effects. The two strains work synergistically through multiple pathways, including ammonia production, bicarbonate secretion, and activation of soil enzyme systems, to increase soil pH and reduce exchangeable aluminum and hydrolyzable acid content. This demonstrates significantly better effects than single-strain methods in improving acidified soil, enhancing soil fertility, and promoting crop growth.

[0026] In one embodiment, the ratio of the effective viable number of Bacillus S1 and Arthrobacter S2 is 1:(1~2).

[0027] The present invention also provides a compound bacterial agent, the active ingredients of which include the bacterial combination described in the above scheme.

[0028] In one embodiment, the total viable count of the active ingredients in the compound microbial agent is not less than 1×10⁻⁶. 8 CFU / g or CFU / mL, further to 1×10 9 CFU / g or CFU / mL.

[0029] The compound microbial agent of the present invention also includes an agriculturally acceptable carrier; the carrier includes, but is not limited to, peat moss, vermiculite, bentonite, fermented organic fertilizer or sterile water.

[0030] The present invention also provides the application of the bacterial combination or the compound bacterial agent described above in at least one of the following: 1) Improve acidified soil; 2) Improve soil fertility; 3) Enhance soil enzyme activity; 4) Promotes crop growth.

[0031] In one embodiment, the pH value of the acidified soil is 4 to 4.9.

[0032] As one implementation method, the improvement of acidified soil includes at least one of increasing soil pH, reducing soil exchangeable aluminum content, reducing soil hydrolyzable acid content, reducing total soil exchangeable acid content, and increasing soil bicarbonate content.

[0033] The bacterial combination of this invention exhibited a synergistic effect of "1+1>2" in pot experiments. Compared with single bacterial treatment or control, this combination significantly increased soil pH (from 4.0 to 6.8), reduced exchangeable aluminum (37%) and hydrolyzable acid (36%), while significantly increasing soil bicarbonate content (60%), thus rapidly and comprehensively improving soil acidity from the root cause.

[0034] In one embodiment, the soil enzyme activity includes at least one of urease, sucrase, protease, and cellulase activities.

[0035] The bacterial combination of this invention can not only directly neutralize acidic substances, but also significantly activate key soil enzyme systems. Experiments show that after inoculation with the bacterial combination, the activities of soil urease, sucrase, protease, and cellulase increased by 199%, 140%, 200%, and 144%, respectively, far exceeding those of single-strain treatments. This indicates that the combination can effectively promote the mineralization and cycling of nutrients such as carbon and nitrogen in the soil, improve the soil biochemical environment, and create a fertile rhizosphere microdomain for crop growth.

[0036] In one implementation, the crop includes corn and / or vegetables; the promotion of crop growth includes increasing at least one of the following indicators: aboveground fresh weight, root fresh weight, plant height, and root length.

[0037] In a pot experiment on maize in acidified soil, the bacterial synthesis of this invention increased the aboveground fresh weight of maize seedlings by 191%, the root fresh weight by 100%, and also significantly promoted plant height and root length. This was attributed to the elimination of soil aluminum toxicity and the improvement of nutrient availability, demonstrating the great potential of this bacterial synthesis in alleviating biotic stress and promoting crop growth.

[0038] The present invention also provides a method for improving acidified soil and promoting crop growth, comprising the following steps: Apply an effective amount of the compound microbial agent described in the above scheme to the soil and / or crop rhizosphere that require improvement.

[0039] The compound microbial agent of the present invention can be combined with conventional agronomic practices, is easy to apply, and is easy to promote on a large scale in acidified farmland.

[0040] As one implementation method, the application method includes furrow application, hole application, drip irrigation, spraying, or seed dressing; the application rate of the compound microbial agent is 10 per acre of total live bacteria containing active ingredients. 12 Up to 10 14 CFU compound microbial agent.

[0041] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides a combination of acid- and alkali-producing bacteria, a compound bacterial agent, and their applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0042] In the embodiments of the present invention, the culture medium involved is specifically as follows: LB enrichment medium: 10.0 g peptone, 5.0 g yeast extract, 10.0 g NaCl, and 1.0 L deionized water; pH adjusted to 5.0 with 1 M HCl. For solid medium, add 15.0 g agar. Sterilize at 121°C for 20 min.

[0043] R2A isolation medium: 0.5 g yeast extract, 0.5 g acid-hydrolyzed casein, 0.5 g peptone, 0.5 g glucose, 0.5 g soluble starch, 0.3 g sodium pyruvate, 0.3 g dipotassium hydrogen phosphate, 0.05 g MgSO4·7H2O, and 1.0 L deionized water. After sterilization, adjust the pH to 4.5 with sterile filtered 1 M HCl solution in a laminar flow hood, and then pour the medium onto the plate.

[0044] Example 1: Systematic Screening, Identification, and Characterization of Acid-Tolerant Alkali-Producing Strains See the roadmap for strain screening and microbial community construction. Figure 1 .

[0045] (1) Sample collection: In order to obtain abundant acid-tolerant and alkali-producing microbial resources, soil samples from the 0-20 cm topsoil layer were collected in Qinggang County, Suihua City, a typical area in Heilongjiang Province with severe soil acidification. The samples were collected from long-term cultivated dry land using the five-point sampling method. After the samples were mixed evenly, they were placed in sterile sampling bags. One part was placed in an ice box and transported back to the laboratory within 4 hours for microbial isolation; the other part was air-dried for basic soil physicochemical property analysis.

[0046] (2) Isolation and initial screening of strains: Weigh 10 g of fresh soil sample and add it to a 250 mL Erlenmeyer flask containing 90 mL of sterile physiological saline and an appropriate amount of glass beads. Shake at 28℃ and 180 rpm for 30 min to prepare a soil homogenate. After standing for 10 min, take the supernatant and perform a 10-fold serial dilution. Take 100 μL of each of the 10-fold serial dilutions. -3 10 -4 10 -5Three dilutions of bacterial culture were spread onto R2A plates at pH 4.5, with three replicates for each dilution. The plates were incubated upside down at 28°C for 3–7 days. Daily observations were made, recording the appearance of different colonies. After incubation, based on differences in colony morphology (size, color, edge, transparency, elevation, etc.), single colonies with different morphologies were picked from plates still showing growth at the highest dilution and streaked onto fresh R2A plates for purification until pure cultures were obtained. All purified strains were inoculated into 96-well plates containing LB agar and incubated at 28°C for 24–48 h. Then, 100 μL of sterile phenol red indicator (color change at pH 6.8–8.2, yellow → red) was added to each well, and the color change was observed within 30 min. Strains whose surrounding medium turned red were selected and preliminarily identified as alkali-producing strains for further screening.

[0047] (3) Quantitative rescreening of acid tolerance and alkali production ability: The alkali-producing strains obtained from the initial screening were inoculated into LB liquid medium (pH 7.0) for activation. 5% (v / v) of the activated bacterial solution was inoculated into LB liquid medium (adjusted with HCl) with initial pH of 4.0, 5.0, and 6.0, respectively, and cultured at 28℃ and 180 rpm for 72 h. The pH values ​​at 0 h and 72 h of culture were measured using a precision pH meter (Mettler Toledo, Switzerland). Simultaneously, the OD value of the culture medium was measured at 600 nm to evaluate the growth of the strains under different acidities. The screening criterion was: the ability to still grow at pH 4.0 (OD value of 4.0). 600 Strains with a pH >0.2 and capable of raising the pH of the culture medium by at least 1.0 unit are defined as acid-tolerant alkali-producing strains. Through this step, approximately 30 potential acid-tolerant alkali-producing strains were screened out from over 100 initially screened strains.

[0048] (4) Classification and identification of strains: 16S rDNA identification was performed using universal prokaryotic 16S rDNA primers 27F (5'-agagtttgatcmtggctcag-3', SEQ ID NO.3) and 1492R (5'-ggytaccttgttacgactt-3', SEQ ID NO.4) for PCR amplification (see the patent "A Method for Rapid Extraction of Total Soil DNA in Small Quantities" (Patent No.: 2005101205847, Publication Date: July 4, 2007)) and sequencing. The results were then compared with the international NCBI GenBank (www.ncbi.nlm.nih.gov) nucleotide database, showing 99% nucleotide homology. These two strains exhibited excellent synergistic effects in subsequent co-pairing experiments and were therefore identified as the core strains of this invention. The 16S rDNA of strains S1 and S2 are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0049] The physiological and biochemical analyses of strains S1 and S2 are shown in Table 1. Table 1. Identification of physiological and biochemical indicators of strains S1 and S2

[0050] SEM images of the strains screened in Example 1 of this invention are shown below. Figure 2 .

[0051] (5) Growth and alkali production kinetics of the strains: To gain a deeper understanding of the characteristics of the core strains S1 and S2, growth and alkali production kinetics were analyzed. S1 and S2 were inoculated into LB liquid medium at pH 5.0 and cultured at 28°C with shaking at 180 rpm. Samples were taken every 6 h to measure OD. 600 The pH of the culture medium was also measured. Simultaneously, a portion of the culture medium was filtered through a 0.22 μm filter membrane, and the ammonium nitrogen (NH4+) in the filtrate was determined using the Berthelot colorimetric method. + The ammonia production capacity was characterized by the concentration of ammonium nitrogen (AM) in the ammonia-nitrogen (AM) content. Growth curves, pH change curves, and ammonium nitrogen accumulation curves were plotted. The results showed that both strains could enter the logarithmic growth phase within 24–36 h, accompanied by a continuous increase in pH and accumulation of ammonium nitrogen, confirming that their alkali production process was closely related to their own growth and metabolism.

[0052] The methods for preserving microbial strains are as follows: Bacillus S1 and Arthrobacter S2 were cultured on 1 / 10 LB liquid or solid medium at 28°C, and could be stored for a short period at 4°C after culture.

[0053] For long-term preservation, glycerol cryovials or freeze-dried tubes are suitable for preserving the strains (for specific methods, please refer to: Zhao Bin, He Shaojiang, Microbiology Experiment, First Edition, Science Press, Beijing, 2002: pp. 202-205).

[0054] Example 2: Evaluation of the effect of the bacterial combination of Bacillus S1 and Arthrobacter S2 on the improvement of the physicochemical properties of acidified soil (pot simulation experiment). The soil samples were collected from typical acidified farmland in Suihua City, Heilongjiang Province, and the soil type was black soil. The basic soil properties were: pH 4.9, organic matter 30.2 g / kg, available nitrogen 105.3 mg / kg, available phosphorus 20.1 mg / kg, available potassium 132.5 mg / kg, and exchangeable aluminum 0.95 cmol / kg. The soil was air-dried and then sieved through a 2 mm sieve for later use.

[0055] The experiment included four treatments: ①CK: control inoculated with an equal volume of sterile water; ②S1: inoculated with a single-strain Bacillus S1 inoculum; ③S2: inoculated with a single-strain Arthrobacter S2 inoculum; ④Bacillus S1 + Arthrobacter S2: inoculated with a compound inoculum of Bacillus S1 and Arthrobacter S2 at a viable count ratio of 1:1. Each treatment was replicated in triplicate, using a completely randomized block design. Plastic pots were used, each containing 2.0 kg of sieved soil sample.

[0056] The preserved Bacillus S1 and Arthrobacter S2 strains were activated on LB agar plates, and single colonies were picked and inoculated into LB liquid medium. The culture was then incubated at 28°C and 180 rpm with shaking until the late logarithmic growth phase (OD2). 600 ≈ 1.0). Under aseptic conditions, the bacterial cells were collected by centrifugation at 5000 rpm for 10 min, washed twice with sterile 0.85% NaCl solution, and finally resuspended in sterile physiological saline to prepare a solution with a concentration of 1×10⁻⁶. 9 A single-strain bacterial suspension at CFU / mL. The compound inoculum is prepared by mixing equal volumes of S1 and S2 bacterial suspensions. For inoculation, use 10 mL of bacterial suspension per kilogram of soil (i.e., 10...). 7 Apply the dosage of CFU / g soil, spray each treatment agent or sterile water evenly onto the soil surface, and mix thoroughly to ensure uniform contact between the bacteria and the soil.

[0057] After inoculation, the soil moisture content of all pots was adjusted to 70% of field capacity using deionized water, and the pot openings were covered with sealing film to reduce water evaporation. The potted plants were then placed in an artificial climate chamber with the following conditions: temperature 25±2℃, relative humidity 65%~75%, and simulated natural light (photoperiod 12 h / 12 ​​h). During the cultivation period, water was added every 3 days using a weighing method to maintain stable soil moisture. The total cultivation time was 15 days.

[0058] After cultivation, soil samples from the 0-10 cm soil layer were collected at multiple points in each pot using a soil auger. After being mixed thoroughly, the samples were divided into two portions. One fresh sample was immediately used for pH and microbial biomass determination, while the other sample was air-dried, ground, and passed through 1 mm and 0.15 mm sieves for chemical property analysis.

[0059] The results of the soil acidity index measurement are shown in Table 2.

[0060] Table 2 Changes in soil pH / latent acidity and bicarbonate content after inoculation with Alkali-producing bacteria.

[0061] The results showed that: (1) All inoculation treatments significantly increased soil pH, with the S1+S2 treatment showing the most significant effect, raising the pH to 6.80, close to the neutral range. (2) The S1+S2 treatment had the most prominent effect on reducing exchangeable acid, with the total exchangeable acid and exchangeable aluminum content decreasing by 39.9% and 37.1% respectively compared to the control (CK), significantly better than the two single-strain treatments. (3) Hydrolyzable acid also showed the same trend, with the S1+S2 treatment showing the largest decrease. (4) Soil HCO3... - The content increased after inoculation, with the S1+S2 treatment showing an increase of 60.2%. These data fully demonstrate that the S1 and S2 strains have a strong synergistic effect in improving soil acidity (including active and latent acids).

[0062] The results of soil enzyme activity assays are shown in Table 3.

[0063] Table 3. Changes in soil enzyme activity after inoculation with Alcaligenes.

[0064] The results showed that: (1) Inoculation with alkali-producing strains, especially the S1+S2 treatment, significantly increased the activities of urease, sucrase, protease, and cellulase in the soil. Among them, the activities of urease, sucrase, protease, and cellulase in the S1+S2 treatment were 199%, 140%, 200%, and 144% higher than those in the control (CK), respectively, which far exceeded the single-strain treatment, demonstrating a significant synergistic effect. The activation of these enzymes means that the decomposition and transformation of organic nitrogen and carbohydrates in the soil were greatly accelerated, and the availability of nutrients was improved. (2) The activity of alkaline phosphatase was slightly increased under the S1+S2 treatment, but not significantly. (3) The activity of catalase decreased in all inoculation treatments, with the most significant decrease in the S1+S2 treatment. Catalase is usually related to the removal of hydrogen peroxide in the soil. Its decrease in activity may be related to changes in the metabolic pathways of soil microorganisms and changes in the level of oxidative stress after inoculation, and may also indicate that the soil redox state is adjusting towards a direction more conducive to nutrient transformation.

[0065] The common maize variety "Zhengdan 958," which is sensitive to aluminum oxide, was selected as the indicator crop. Seeds were surface-sterilized with a 5% sodium hypochlorite solution for 10 min, thoroughly rinsed with sterile water, and then placed in petri dishes lined with moistened filter paper. Germination was carried out in the dark at 25℃ until the radicle emerged (radicle length approximately 1-2 mm). Three germinated seeds were sown per pot at a depth of approximately 2 cm. After emergence (approximately 5 days), each pot was thinned to produce one robust seedling with uniform growth. The cultivation conditions were the same as in Example 2 (temperature 25±2℃, photoperiod 12h / 12h, maintaining field capacity at 70%). The growth cycle was 15 days.

[0066] After cultivation, carefully remove each corn plant along with its root system, immerse it in a tray of water, and gently rinse to remove any attached soil. For a comparison of the growth-promoting abilities of single-strain and synthetic microbial communities in soil simulation experiments, see [link to relevant documentation]. Figure 3 After blotting the surface moisture with filter paper, the following measurements were performed: (1) Morphological indicators: Use a ruler to measure plant height (from stem base to highest leaf tip) and root length (length of longest taproot).

[0067] (2) Biomass indicators: The plants were separated into aboveground parts and roots at the root collar, and their fresh weight was measured separately using an electronic balance. Subsequently, the samples were placed in kraft paper bags and placed in an oven at 105℃ for 30 min to kill the enzymes, and then dried at 75℃ to constant weight (about 48~72 h), and the dry weight of the aboveground parts and roots was measured. The results of the measurement of maize seedling growth indicators are shown in Table 4.

[0068] Table 4. Changes in maize growth 15 days after inoculation with Alcaligenes.

[0069] The results showed that: (1) Compared with the control (CK), all inoculation treatments significantly promoted the growth of maize seedlings, but the S1+S2 treatment had an overwhelming advantage. The aboveground fresh weight and dry weight were 2.9 times and 3.0 times that of the CK, respectively, and the root fresh weight and root dry weight were 2.0 times and 2.1 times that of the CK, respectively. (2) In terms of morphology, the plant height and root length of the S1+S2 treatment were also significantly better than other treatments, increasing by 83.3% and 41.6% respectively compared with the CK. (3) Although the single-strain treatments (S1, S2) also had a growth-promoting effect, all their indicators were significantly lower than those of the S1+S2 treatment. This directly demonstrates the excellent synergistic ability of the S1 and S2 strain combination in relieving the inhibition of crop growth by acidified soil and significantly stimulating the growth potential of crops. This growth-promoting effect is a direct biological manifestation of the comprehensive effects of the aforementioned soil acidity improvement, aluminum toxicity reduction, soil enzyme activity enhancement, and nutrient supply improvement.

[0070] Based on the results of the above embodiments, combined with the results of strain S1 ( Bacillus sp .) and S2 ( Arthrobacter sp The known physiological characteristics of .) and their synergistic mechanisms include the following levels: (1) Direct alkali production and neutralization of reactive acids: Both strains have strong acid resistance and alkali production (such as ammonia production) capabilities. In the soil, they produce NH3 through metabolic activities (such as urease hydrolysis of urea, amino acid deamination, etc.), and NH3 dissolves in water to form NH4. + and OH - Directly neutralizes H in the soil solution + This rapidly raises the pH level. Simultaneously, HCO3 may be produced during the metabolic process. - Buffer substances, consuming H +(HCO3) - + H + → H2O + CO2), further enhancing the soil's buffering capacity.

[0071] (2) Passivating aluminum ions and reducing latent acidity: Increased soil pH directly leads to the release of highly toxic soluble Al ions. 3+ The conversion can lead to low-toxicity or non-toxic aluminum hydroxyl polymers and precipitated aluminum. Furthermore, bacterial cell walls or secreted extracellular polymers (EPS) are rich in anionic groups such as carboxyl and phosphate groups, which can fix Al through complexation and adsorption. 3+ This further reduces the exchangeable aluminum content and mitigates the damage of aluminum toxicity to the root system.

[0072] (3) Secretion of enzymes and metabolites, activating soil fertility: As shown in Example 3, the strain combination significantly enhanced the activity of various soil enzymes. This may be due to: (a) the strains themselves secreting these enzymes; (b) the introduction of the strains altering the rhizosphere microbial community structure and stimulating the activity of native beneficial microorganisms; and (c) substances produced by the strains' metabolism (such as plant hormones, vitamins, organic acids, etc.) stimulating the metabolism of plant roots and soil microorganisms. The increased activity of urease and protease accelerated the mineralization of organic nitrogen; the increased activity of sucrase and cellulase promoted the decomposition of carbon sources, providing more effective nutrients and energy for microorganisms and plants.

[0073] (4) Improves the rhizosphere microenvironment, directly or indirectly promoting growth: Through the combined effects of the above, the microbial agent creates an excellent rhizosphere environment for crop roots that is "low in acid, low in aluminum, and high in fertility." Root development is promoted (root length and root weight increase), enhancing its ability to absorb water and nutrients. In addition, Bacillus and Arthrobacter usually have the characteristics of producing plant growth hormones (such as IAA), siderophores, and antagonizing pathogens, which can produce direct biostimulation and bioprotection effects on plant growth.

[0074] In summary, the acid-tolerant and alkali-producing strain combination S1 and S2 provided by this invention achieves a combination of "treating the symptoms" (rapidly adjusting acidity) and "treating the root cause" (reducing latent acidity and activating the ecosystem) in acidified soil through the synergistic effect of multiple pathways and multiple targets, ultimately significantly promoting crop growth and demonstrating great application potential.

[0075] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A bacterial assemblage, characterized in that, Including Bacillus ( Bacillus sp. S1 and Arthrobacter ( Arthrobacter sp S2; the preservation number of the Bacillus S1 is CGMCC No. 29201; the preservation number of the Arthrobacter S2 is CGMCC No. 29202.

2. The bacterial assemblage according to claim 1, characterized in that, The ratio of the effective viable counts of Bacillus S1 and Arthrobacter S2 is 1:(1~2).

3. A compound microbial agent, characterized in that, The active ingredient comprises the bacterial combination as described in claim 1 or 2.

4. The compound microbial agent according to claim 3, characterized in that, The total viable bacteria count of the active ingredients in the compound microbial agent is not less than 1×10⁻⁶. 8 CFU / g or CFU / mL.

5. The use of the bacterial combination of claim 1 or 2 or the compound bacterial agent of claim 3 or 4 in at least one of the following: 1) Improve acidified soil; 2) Improve soil fertility; 3) Enhance soil enzyme activity; 4) Promotes crop growth.

6. The application according to claim 5, characterized in that, The improvement of acidified soil includes at least one of the following: increasing soil pH, reducing soil exchangeable aluminum content, reducing soil hydrolyzable acid content, reducing total soil exchangeable acid content, and increasing soil bicarbonate content.

7. The application according to claim 5, characterized in that, The soil enzyme activity includes at least one of urease, sucrase, protease and cellulase activities.

8. The application according to claim 5, characterized in that, The crop includes corn and / or vegetables; the promotion of crop growth includes increasing at least one of the following indicators: aboveground fresh weight, root fresh weight, plant height, and root length.

9. A method for improving acidified soil and promoting crop growth, characterized in that, Includes the following steps: Apply an effective amount of the compound microbial agent as described in claim 3 or 4 to the soil and / or crop rhizosphere that require improvement.

10. The method according to claim 9, characterized in that, The application methods include furrow application, hole application, drip irrigation, spraying, or seed dressing; the application rate of the compound microbial agent is 10 per acre of total live bacteria containing active ingredients. 12 Up to 10 14 CFU compound microbial agent.

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