Micro-ecological restoration liquid complex microbial inoculant suitable for saline-alkali soil and biological bacterial fertilizer thereof

Abstract

The invention relates to the field of microbial agents, in particular to a micro-ecological restoration liquid composite microbial agent suitable for saline-alkali soil and a biological bacterial fertilizer of the micro-ecological restoration liquid composite microbial agent. The micro-ecological restoration liquid complex microbial inoculant suitable for the saline-alkali soil is prepared from restoration salt-tolerant bacteria, namely bacillus subtilis, nocardia halospora, nitrogen-fixing bacteria, namely azotobacter chroococcum, phosphate solubilizing bacteria, namely pseudomonas fluorescens, potassium solubilizing bacteria, namely bacillus mucilaginosus, growth-promoting bacteria, namely azospirillum brasilense, biocontrol bacteria, namely bacillus megatherium and synergistic bacteria, wherein the restoration salt-tolerant bacteria are bacillus subtilis, nocardia halospora, the nitrogen-fixing bacteria are azotobacter chroococcum, the phosphate solubilizing bacteria are pseudomonas fluorescens, the potassium solubilizing bacteria are bacillus mucilaginosus, the growth-promoting bacteria are azospirillum brasilense, the biocontrol bacteria are bacillus megatherium, and the synergistic bacteria are prepared from bacillus subtilis The biological bacterial fertilizer for the saline-alkali soil is prepared from the liquid complex microbial inoculant, compared with a conventional biological bacterial fertilizer, the adaptability to the saline-alkali soil is greatly improved, the saline-alkali soil is effectively improved, and the crop yield is remarkably increased.

Description

Technical Field

[0001] This invention relates to the field of microbial agents, specifically to a microecological restoration liquid compound microbial agent and its bio-fertilizer suitable for saline-alkali land. Background Technology

[0002] Saline-alkali land is rich in sodium salts (Na) due to improper irrigation, tidal intrusion, or natural geographical background. + ) and carbonates (CO3) 2- / HCO 3- Saline-alkali soils contain alkaline substances such as sodium, leading to high pH, ​​high electrical conductivity (EC value), and high sodium adsorption ratio (SAR value). These properties result in soil compaction and poor permeability, causing severe osmotic stress and ion toxicity, inhibiting plant absorption of water and nutrients, and causing deficiencies in essential elements such as calcium, potassium, and zinc, leading to physiological drought, nutrient imbalance, and stunted growth in crops. Furthermore, saline-alkali lands are often accompanied by low organic matter content, poor microbial activity, and inhibited enzyme systems, further limiting soil fertility and ecological functions. From an ecological perspective, abandoning saline-alkali land is a passive approach. Exposed surfaces easily exacerbate salt accumulation, wind erosion, and the upward flow of salt from groundwater, leading to the expansion of secondary salinization, pollution of surrounding water bodies and soil, and spillover ecological risks. Conversely, scientific vegetation restoration and cultivation management can inhibit salt rise and gradually improve the micro-ecological environment through biological drainage and organic matter enhancement.

[0003] Under the premise of ensuring ecological security, improving crop productivity and resource utilization efficiency in saline-alkali land has become a key research focus. Direct application of conventional fertilizers not only significantly reduces fertilizer efficiency due to high pH and ionic strength, but also exacerbates secondary soil salinization and compaction, further worsening the root growth environment. In recent years, using microbial agents to improve saline-alkali soil, enhance crop salt tolerance, and reduce fertilizer use has become a research and application hotspot. Therefore, developing a compound microbial agent suitable for saline-alkali land to regulate soil microecology, promote nutrient activation, and improve physical structure through biological means has become an urgent need for the green development of agriculture in saline-alkali land. Although some existing microbial agents have been attempted for saline-alkali land improvement, their application effects are still unsatisfactory, with significant bottlenecks: the high osmotic pressure, high pH, ​​and ionic toxicity of saline-alkali soil constitute an extreme stress environment. Exogenous agents not only face direct physiological inhibition but also need to compete for nutrients and ecological niches with more resilient native microorganisms. Furthermore, the drastic fluctuations in soil moisture, temperature, and organic matter content make it difficult for inoculated strains to colonize and function. Secondly, single-strain microorganisms have limited functions, only able to alleviate certain types of stress (such as phosphorus solubilization, acid production, and synthesis of osmotic regulators), and cannot systematically address the complex environment of saline-alkali land with multiple intertwined stresses. Their improvement process is slow and the effects are unstable; the metabolic activities of some strains may even temporarily increase local pH or EC values, exacerbating salinity problems. These factors combined make the effects of existing microbial agents difficult to predict and replicate, failing to meet the stability and large-scale requirements of agricultural production in saline-alkali land.

[0004] Compared to single-strain inoculants, compound microbial agents offer the core advantage of synergistic effects through multiple strains, achieving functional complementarity, enhanced environmental adaptability and stability, and improved system repair capabilities. However, their industrial production faces significant challenges: Separate fermentation of each strain results in complex processes, high equipment investment, and high energy consumption; mixed fermentation, due to differences in the requirements of different strains for culture medium components (carbon-nitrogen ratio, trace elements, pH) and fermentation conditions (temperature, dissolved oxygen, stirring rate), easily leads to uneven growth rates—fast-growing strains dominate, competing for nutrients and producing inhibitory substances (such as antibiotics and organic acids), causing slow-growing strains to be suppressed or even die, resulting in an imbalanced microbial community structure, functional degradation, and even off-flavors or toxins in the final product, severely affecting product consistency and safety. While existing technologies employ a series of control strategies, such as optimizing culture medium formulations, segmented control of fermentation parameters, symbiotic acclimatization of strains, and optimization of inoculation ratios, these methods do not address how to better suit industrial production. Therefore, the methods employed are relatively simplistic and cannot comprehensively optimize various conditions while considering the complex characteristics of each strain. Meanwhile, existing compound microbial agents suitable for saline-alkali land still have problems such as unstable efficacy, short shelf life, and large amount of fertilizer required after fertilization. Summary of the Invention

[0005] To address the challenges of low crop yields in saline-alkali lands due to their high pH, ​​high electrical conductivity (EC value), and high sodium adsorption ratio (SAR value), the poor effectiveness of conventional fertilizers, and their potential to further deteriorate soil properties, existing single or compound microbial agents are insufficient to meet the needs of saline-alkali lands. Furthermore, the industrial preparation of compound microbial agents is prone to issues such as microbial imbalance, low yield, and difficulty in controlling product quality to optimize various conditions. This invention provides a microecological restoration liquid compound microbial agent and its bio-fertilizer suitable for saline-alkali lands, thus solving the aforementioned problems.

[0006] The technical solution of the present invention is as follows:

[0007] This invention provides a microecological remediation liquid compound microbial agent suitable for saline-alkali land, characterized by comprising the following eight functional strains prepared by electrochemical low-hertz water through three-stage sequential inoculation and fermentation: Salt-tolerant bacteria: Bacillus subtilis, Nocardiopsis halotolerans; Nitrogen-fixing bacteria: Azotobacter chroococcum; Phosphate-solubilizing bacteria: Pseudomonas fluorescens; Potassium-solubilizing bacteria: Paenibacillus mucilaginosus; Growth-promoting bacteria: Azospirillum brasilense; Biocontrol bacteria: Pseudomonas putida; and Synergistic bacteria: Trichoderma asperellum.

[0008] S1: Propagation of salt-tolerant and nitrogen-fixing bacteria: Add electrolytic low-hertz water to the sterilized main fermenter, and add pre-mixed Bacillus subtilis, Haloxylon ammodendron, and Azotobacter spp. Adjust the fermentation conditions to the first-order fermentation conditions: pH=7.5±0.3, dissolved oxygen 25-35%, temperature 32℃, humidity of the top space of the fermenter 90-92%, fermentation time 34-38 hours;

[0009] S2: Introduction of phosphate-solubilizing bacteria, growth-promoting bacteria, and biocontrol bacteria: After the S1 stage, the conditions are adjusted to the second fermentation sequence, and pre-mixed seed liquids of *Pseudomonas fluorescens*, *Azotobacter brasiliensis*, and *Bacillus megaterium* are added to the main fermenter. The second fermentation sequence conditions are: pH=7.8±0.3, dissolved oxygen 30-40%, temperature 32℃, humidity of the top space of the fermenter 91-93%, and fermentation time 16-20 hours.

[0010] S3: Initiate the synergistic metabolism of potassium-solubilizing bacteria and Trichoderma, and supplement with salt-tolerant bacteria: After the fermentation of the S2 stage is completed, after Bacillus mucilaginosus seed liquid is introduced into the main fermenter and fermented for 12 hours, the fermentation parameters are detected (pH should drop to 6.5-7.0, dissolved oxygen >50%). Bacillus subtilis seed liquid is introduced, and mechanical stirring (200-300 rpm) is started immediately. Dissolved oxygen is maintained at 60%, temperature at 28℃. After fermentation for 12 hours, Trichoderma echinococcosis seed liquid is introduced to enter the fungal fermentation stage, and the fermentation conditions are adjusted to the third time sequence: pH=7.5±0.3, dissolved oxygen 40-450%, temperature at 32℃, humidity of the top space of the fermenter at 85-88%, fermentation for 40-44 hours;

[0011] When the number of viable bacteria in the main fermenter is ≥2×10 10 Fermentation ends when CFU / mL is reached.

[0012] Preferably, the ratio of the repair bacteria to halophilic bacteria: nitrogen-fixing bacteria: phosphorus-solubilizing bacteria: potassium-solubilizing bacteria: growth-promoting bacteria: biocontrol bacteria: synergistic bacteria is 3:2:1:1:1.5:1:0.5, and the internal ratio of the repair bacteria to halophilic bacteria is Bacillus subtilis: Haloxylon ammodendron = 2:1.

[0013] Preferably, the seed culture preparation method is as follows: eight functional bacterial strains are inoculated from the preservation slant onto a special slant culture medium and cultured at 30°C for 24-48 hours until the logarithmic growth phase (OD50). 600 When the concentration is ≥1.0, the samples were transferred to shake flasks for culture, and then transferred to eight primary seed tanks for independent culture.

[0014] The method for preparing the premixed seed solution is as follows: when the bacterial count in the primary seed tank is ≥1×10⁻⁶. 9 At CFU / mL, Bacillus subtilis (logarithmic growth phase (8-12h) culture), Haloxylon ammodendron, and Azotobacter chrysogenum were mixed in a ratio of 2:1:2 to obtain premixed seed liquid 1. Pseudomonas fluorescens, Azotobacter brasiliensis, and Bacillus megaterium seed liquid were mixed in a volume ratio of 2:3:2 to obtain premixed seed liquid 2. After incubation at 30℃ for 30 minutes, the two premixed seed liquids were used for sequential fermentation.

[0015] When the number of Bacillus mucilaginosus and Bacillus subtilis bacteria in the primary seed tank is ≥1×10 9 At CFU / mL, the number of Trichoderma acicularis spores is ≥1×10⁻⁶. 7 At a concentration of spores / mL, it can be used for sequential fermentation;

[0016] The *Bacillus subtilis* was cultured on LB medium supplemented with 0.3% NaCl, the *Haloxylon ammodendron* on ISP2 medium supplemented with 0.5% NaCl, nitrogen-fixing bacteria and growth-promoting bacteria on Asbestos medium, phosphate-solubilizing bacteria on KB medium, biocontrol bacteria on LB medium, potassium-solubilizing bacteria on potassium-solubilizing medium, and synergistic bacteria on PDA medium; all seven media were prepared using electrolytic low-Hertz water and sterilized at 121°C for 20 minutes.

[0017] The inoculation amounts of the premixed seed solution 1, premixed seed solution 2, Bacillus mucilaginosus seed solution, Trichoderma echinosporum seed solution, and Bacillus subtilis seed solution (culture in the early stage of the stable period (18-24h)) are 11.8%-12.2%, 6.8%-7.2%, 2.8%-3.2%, 2.8%-3.2%, and 2.8%-3.2% of the fermentation volume, respectively. The inoculation amount of electrochemical low-hertz water is 71.4%-72.6% of the fermentation volume. The electrochemical low-hertz water has a molecular cluster of ≤6 molecules and a pH of 7.0-7.5.

[0018] Preferably, during the S1 stage, when fermentation reaches the middle of the logarithmic growth phase, growth factors betaine, sodium citrate, and sodium chloride are added; after the S1 stage fermentation ends and the system pH stabilizes at 7.8±0.2, growth factors calcium phytate, L-proline, and MnSO4 are added; after the S3 stage is started by inoculating potassium-solubilizing bacteria, growth factors potassium feldspar powder, humic acid, and ZnSO4 are added.

[0019] Preferably, in stage S1, betaine, sodium citrate, and sodium chloride are added in batches using a pulse addition method: the final concentration of betaine is 0.04% (w / v), the final concentration of sodium citrate is 0.15% (w / v), and the final concentration of sodium chloride is 0.3% (w / v); the addition is done in 3 batches, with an interval of 3 hours between each addition, and each addition is completed within 10 minutes; the addition order is: first add sodium chloride, then add betaine and sodium citrate simultaneously 30 seconds later;

[0020] In stage S2, calcium phytate was added continuously at a flow rate of 0.15 g / L·h to a final concentration of 1.8 g / L; L-proline and MnSO4 were added simultaneously at a flow rate of 0.002 g / L·h to final concentrations of 0.03% (w / v) and 0.005% (w / v), respectively. One hour after the calcium phytate addition was started, the addition of L-proline and MnSO4 was then initiated.

[0021] In stage S3, potassium feldspar powder, humic acid, and ZnSO4 were added in batches and via atomization: the potassium feldspar powder had a particle size of 200 mesh and a total concentration of 10 g / L, added in two batches of 5 g / L each, with an interval of 5 hours; the final concentrations of humic acid and ZnSO4 were 0.2% (w / v) and 0.003% (w / v), respectively, and were added via simultaneous atomization with droplet diameters of 10-15 μm, starting simultaneously with the second addition of potassium feldspar powder; the potassium feldspar powder was added first, and the atomization addition of humic acid and ZnSO4 began 30 minutes later.

[0022] This invention also provides a micro-ecological restoration bio-fertilizer suitable for saline-alkali land, which is prepared using the above-mentioned liquid compound microbial agent suitable for saline-alkali land soil micro-ecological restoration.

[0023] Preferably, the preparation method includes:

[0024] A1: The liquid compound microbial agent, electrochemical low-hertz water, and solid components (lignite powder, wheat bran, and soybean meal) are added to the mixing system. After uniform mixing, the mixture is conveyed to the solid-state fermentation system production line via an intelligent material distribution system (patent authorization announcement number CN 116553963B) for the first solid-state fermentation. The solid-state fermentation system automatically adjusts the temperature during the fermentation process through a temperature sensor linked to the intermittent micro-burst oxygen frequency: the initial stage temperature is 42-45℃, maintained for 22-24 hours; the peak stage temperature is 60-65℃, maintained for 46-48 hours; and the final stage temperature is 40-45℃, maintained for 22-24 hours.

[0025] A2: The material obtained from the first solid-state fermentation in A1 is rearranged into the aging and fermentation production line through an intelligent material distribution system for a second aging and fermentation: the naturally piled material is deeply decomposed using the residual heat and residual microorganisms. After 10-12 days, the fermentation ends, and the bio-fertilizer suitable for saline-alkali land is obtained.

[0026] Preferably, the total amount of the solid components lignite powder, wheat bran, and soybean meal in A1 is 100%, and the proportions of the three components are: lignite powder 70-85%, wheat bran 8-15%, and soybean meal 8-20%. The particle size of the lignite powder is 80-100 mesh, and the initial moisture content is 10-15%. The initial moisture content of the wheat bran and soybean meal is 8-12%. After the above solid components are mixed evenly, electrochemical low-hertz water is added and stirred thoroughly. At the same time, liquid compound microbial agent is added simultaneously during the stirring process, and the amount added is calculated as 0.8-10% of the total amount of solid components, so that the total moisture content of the mixture reaches 50-60%.

[0027] Preferably, the sign that the first fermentation of stage A1 has ended is: the temperature is maintained at the final stage temperature for 24 consecutive hours, the material humidity is ≤40%, the material color is dark brown, there is no odor, it is loose when squeezed, and there is no sticking.

[0028] Preferably, the second fermentation in stage A2 is marked as follows: when the material naturally cools down to a moisture content of 15-29%, has a uniform dark brown color, a loose texture, an earthy smell without other odors, a pH of 6.5-7.5, and a beneficial live bacteria count ≥200 million / g, the second fermentation is complete.

[0029] The beneficial effects of this invention are as follows:

[0030] This invention provides a microecological restoration liquid compound microbial agent suitable for saline-alkali land. Through a "three-stage sequential fermentation and dynamic regulation" design in the main tank, salt-tolerant, nitrogen-fixing, phosphorus-solubilizing, potassium-solubilizing, growth-promoting, biocontrol, and synergistic strains are organically combined according to function and time sequence. By precisely controlling dissolved oxygen, pH, temperature, humidity, and nutrient addition strategies at each stage, the eight functional bacteria are ensured to reproduce normally and function efficiently under high salt stress. This solves the ecological restoration problems of saline-alkali land caused by high salt, high pH, ​​nutrient fixation, and low microbial activity. Haloxylon ammodendron directly reduces soil conductivity through specific metabolism, while Bacillus subtilis secretes an osmotic protectant to enhance the overall salt tolerance of the bacterial community. The two work synergistically to reduce salt and resist stress, providing a protective environment for nitrogen-fixing bacteria and other non-fermenting bacteria. Salt-tolerant functional bacteria provide the foundation for survival, solving the problem of low bacterial survival rate under high salt stress in saline-alkali land. *Azotobacter chrysogenum* activates nitrogenase in a low-salt microenvironment, continuously providing an organic nitrogen source. *Pseudomonas fluorescens* and *Bacillus mucilaginosus* activate phosphorus and potassium elements fixed by calcium and magnesium ions, respectively. *Azotobacter brasiliensis* secretes IAA to promote root development under salt stress. These three bacteria synergistically construct a nutrient-efficient utilization system, solving the problems of low nutrient availability and inhibited crop growth in saline-alkali land. *Bacillus megaterium* acts as a biocontrol bacterium, effectively controlling common soil-borne diseases in saline-alkali land. *Trichoderma echinosporum*, as a synergistic bacterium, secretes organic acids to assist in alkalinity reduction and mineral decomposition, forming a functional complementarity with salt-tolerant bacteria. This constructs a biocontrol, alkalinity-reducing, and synergistic functional enhancement layer, solving the problems of biological barriers and high pH in saline-alkali land. By employing a three-stage sequential fermentation and dynamic control strategy in the main tank, eight functional bacteria are inoculated and activated stepwise. This allows the liquid compound bacterial agent to significantly enhance the colonization capacity and metabolic stability of the bacterial community in a high-salt environment, while maintaining comprehensive repair functions. It also demonstrates high adaptability to the ecological restoration of saline-alkali land. This approach solves the problems of salt-tolerant bacteria being inhibited, nitrogenase inactivation, and failure of phosphorus and potassium solubilization functions, as well as the collapse of the fermentation system, caused by differences in bacterial growth rates, metabolic antagonism, and pH compatibility conflicts during synchronous inoculation.

[0031] This invention groups eight functional bacteria according to their growth requirements and performs time-sequential culture:

[0032] The first inoculation sequence consists of halophilic Bacillus subtilis, Halophytum halophilicum, and nitrogen-fixing bacteria Azotobacter chrysogenum. These three bacteria work synergistically to reduce salt content, fix nitrogen, and provide stress resistance. The fermentation conditions for this sequence are set as follows: dissolved oxygen 25-35%, satisfying the low-oxygen protection required for nitrogenase activity while also accommodating the basic respiratory needs of halophilic bacteria; pH 7.5±0.3, matching the optimal activity range for nitrogen-fixing bacteria and promoting efficient expression of Bacillus subtilis osmotic regulation genes; constant temperature fermentation at 32℃ for 36 hours, balancing the slow growth of Halophytum halophilicum and the rapid colonization of Bacillus subtilis, preventing nitrogenase inactivation due to high temperatures; and a humidity of 90-92% in the top space of the fermenter, effectively maintaining moisture balance in a high-salt environment and preventing increased osmotic stress due to water evaporation. The three bacteria undergo mixed fermentation to form a complementary function: Haloxylon ammodendron directly reduces the system's conductivity through metabolism, Bacillus subtilis secretes an osmotic protectant to enhance overall salt tolerance, and the two work together to create a low-salt microenvironment for nitrogen-fixing bacteria; Azotobacter chrysogenum continues to fix nitrogen after activation, providing a key nitrogen source for subsequent bacterial communities.

[0033] The second inoculation sequence includes *Pseudomonas fluorescens*, *Azospirillum brasiliensis*, and *Bacillus megaterium*. These three bacteria synergistically exert their functions of phosphorus solubilization, growth promotion, and biocontrol. The fermentation conditions for this sequence are set as follows: dissolved oxygen 30-40%, supporting the high-aerobic process of phosphorus-solubilizing enzyme synthesis and antimicrobial peptide production; pH 7.8±0.2, optimizing the phytase secretion of *Pseudomonas fluorescens* and the IAA synthesis pathway of *Azospirillum brasiliensis*; fermentation at 32℃ for 18 hours, fully utilizing the nitrogen source and low-salt environment established in the first stage to promote efficient functional metabolism. The humidity in the fermenter top space is controlled at 91-93%, effectively preventing excessive water evaporation under high aeration, avoiding osmotic pressure reversal due to fermentation broth concentration, and ensuring the efficient operation of phytase activity in phosphorus-solubilizing bacteria and the IAA synthesis pathway in growth-promoting bacteria in a weakly alkaline environment. The three bacteria exhibit synergistic metabolic effects: phosphorus activated by phosphorus-solubilizing bacteria serves as a key substrate for IAA synthesis in growth-promoting bacteria, while biocontrol bacteria utilize the same carbon source and grow compatiblely with both, producing antimicrobial peptides that effectively control potential diseases. If this stage is started prematurely, it will lead to the loss of phospholytic enzyme activity, insufficient IAA synthesis, and failure of biocontrol function.

[0034] In the third time sequence, after the *Bacillus mucilaginosus* seed culture is introduced into the main fermenter and fermented for 12 hours, fermentation parameters are monitored (pH should drop to 6.5-7.0, dissolved oxygen >50%). Then, *Bacillus subtilis* seed culture (3% inoculum) is introduced, and mechanical stirring (200-300 rpm) is immediately started. Dissolved oxygen is maintained at 60%, and the temperature at 28°C. After 12 hours of fermentation, *Trichoderma echinococcus* seed culture (3% inoculum) is introduced, initiating the fungal fermentation stage. These three processes constitute a functional enhancement layer of "potassium solubilization-synergy-stabilization". The delayed inoculation of *Trichoderma* in this invention avoids metabolic antagonism between *Trichoderma* and bacteria, while the pre-growth of *Bacillus mucilaginosus* provides an acidic pretreatment environment for *Trichoderma*. The addition of salt-tolerant bacteria effectively controls the systemic salinity rise, ensuring the stability of later fermentation stages. The fermentation conditions for this sequence were adjusted as follows: dissolved oxygen 40-45%, combined with mechanical stirring to meet the strict aerobic characteristics of *Trichoderma*; pH 7.5±0.2, balancing bacterial activity and the organic acid secretion function of *Trichoderma*; fermentation at 32℃ for 42 hours to coordinate the process of *Trichoderma* spore formation and the continuous decomposition of potassium feldspar by potassium-solubilizing bacteria. The humidity in the top space of the fermenter was controlled at 85-88%, precisely matching the low-humidity requirements of *Trichoderma* aerobic fermentation, preventing excessive viscosity of the culture system, and ensuring uniform dispersion of potassium feldspar powder and efficient dissolved oxygen transfer. The three components work synergistically: the organic acids secreted by *Trichoderma echinosporum* work synergistically with *Bacillus mucilaginosus* to decompose potassium minerals, significantly increasing the potassium release rate, while the added halophilic bacteria further strengthen the system's resilience, thus achieving a systematic construction and maximum output of the overall function of the microbial agent. *Bacillus subtilis* in premixed seed solution 1 is mainly responsible for the basic improvement and microenvironment regulation of saline-alkali soil in the early stage; the *Bacillus subtilis* introduced in the third sequence forms a synergistic repair network with *Bacillus mucilaginosus* and *Trichoderma echinosporum*, strengthening the desalination and structural improvement of saline-alkali soil in the later stage. This time-sequential dual access strategy can significantly improve the remediation effect of saline-alkali land.

[0035] The three groupings and corresponding time sequences designed in this invention are scientifically efficient, ensuring no interspecies antagonism during the fermentation process of each strain, that their own propagation is not inhibited, and that the fermentation products of strains propagating earlier do not adversely affect strains propagating later, allowing them to be efficiently utilized by strains propagating later to promote growth. Furthermore, due to the rational design, the secondary propagation process of the liquid compound microbial agent (i.e., the time-sequential propagation in the main fermenter) only requires the addition of trace amounts of growth factors and electrolytic low-hertz water, eliminating the need for additional carbon and nitrogen sources, thus simplifying the production process. In addition, the secondary fermentation of this invention only requires the use of a single independent main fermenter, avoiding idle tanks, reducing the probability of cross-contamination, and effectively lowering production costs.

[0036] In the preparation of the liquid compound microbial agent, electrochemical low-Hertz water is used for humidity control in the top space of the fermenter, inoculum activation, primary culture, and time-sequential culture in the main fermenter. This electrochemical low-Hertz water consists of six water molecules, has a permeability more than 30% higher than ordinary water, strong dissolving power, and can quickly pass through cell membrane water channels (only 0.5 nanometers in diameter) to efficiently carry stress-resistant and functional substrates such as betaine and calcium phytate into the bacterial cells, and accelerate the excretion of metabolic waste under salt stress. Simultaneously, this water can enhance the permeability of microbial cell membranes, promote quorum sensing signal transmission between halophilic bacteria and nitrogen-fixing bacteria, and between phosphate-solubilizing bacteria and growth-promoting bacteria, optimizing the metabolism of the compound microbial community. The electrochemical low-Hertz water is weakly alkaline (pH 7.5-8.0), which can neutralize pH fluctuations caused by Trichoderma acid production and phosphate-solubilizing bacteria secretion of organic acids during fermentation, maintaining the optimal pH environment for enzymatic reactions at each stage (such as nitrogenase, phytase, and silicate enzymes). The EC value of the first-order fermentation broth decreased rapidly, enhancing the expression of the proline synthase gene in Bacillus subtilis and thus improving the overall salt tolerance of the bacterial community. In the fermentation of phosphate-solubilizing bacteria, the electrochemically treated low-Hertz water increased phytase activity by 35% and the effective phosphorus concentration to over 45 mg / L, significantly improving the activation efficiency of insoluble phosphorus in saline-alkali soils. Furthermore, the electrochemically treated low-Hertz water, by optimizing carbon source metabolism and ion chelation effects, promoted the increase of nitrogenase activity in Azotobacter chrysogenum under low-salt conditions, establishing a reliable nitrogen source basis; by enhancing the ability of Pseudomonas fluorescens to secrete organic acids such as citric acid, it effectively dissolved calcium and magnesium-fixed phosphorus in saline-alkali soils; by improving the adsorption and acidolysis efficiency of potassium feldspar by Bacillus mucilaginosus, it increased the decomposition rate; and by promoting the synthesis of IAA by Azotobacter brasiliensis under salt stress, it ensured the crop growth-promoting function. The advantages of electrochemical low-hertz water for complex microbial communities are not only reflected in the functional enhancement of individual bacterial species, but also in the system optimization of the microbial community cascade reaction: the water promotes the salt reduction of salt-tolerant bacteria to create a living space for nitrogen-fixing bacteria, nitrogen-fixing bacteria provide nitrogen to support the reproduction of phosphorus / potassium-solubilizing bacteria, can dynamically buffer pH fluctuations at each stage, maintain a wide suitable growth range for complex microbial communities from neutral to weakly alkaline, reduce metabolic antagonism, and enhance overall stress resistance.

[0037] The microecological restoration liquid compound microbial agent suitable for saline-alkali land achieves a progressive restoration effect of salt reduction and stress resistance, nutrient activation, and functional synergy through the scientific formulation of eight functional bacteria and a three-stage sequential fermentation process.

[0038] Firstly, there's the salt reduction and stress resistance, and the construction of a microenvironment. Bacillus subtilis and Haloxylon ammodendron, as pioneer strains of salt tolerance, rapidly construct low-salt microenvironments by secreting osmotic protectants and through direct metabolism, respectively, thus reducing soil electrical conductivity and providing a foundation for the survival of non-salt-tolerant functional bacteria such as nitrogen-fixing and phosphate-solubilizing bacteria. During the fermentation stage, the two strains work synergistically in a 2:1 ratio, forming a dual salt-resistance barrier of "rapid colonization and continuous salt reduction," successfully overcoming the core challenge of the survival of functional microbial communities under high salt stress in saline-alkali land.

[0039] Secondly, there is the synergistic activation and supply of nutrients. *Azotobacter globosum* activates nitrogenase in a low-salt environment, continuously providing ammonium nitrogen and laying the nutritional foundation. *Pseudomonas fluorescens* secretes organic acids such as citric acid to dissolve calcium and magnesium-fixed phosphorus, while *Bacillus mucilaginosus* releases silicate enzymes to dissociate potassium feldspar. With the assistance of organic acids secreted by *Trichoderma*, both form a synergistic effect of phosphorus and potassium solubilization and alkalinity reduction, significantly improving the availability of phosphorus and potassium in saline-alkali soils. *Azotobacter brasiliensis* synthesizes IAA under salt stress to promote root development, working together with nitrogen-fixing bacteria to perform nitrogen fixation and growth-promoting functions, enhancing crop salt tolerance and nutrient absorption capacity.

[0040] Thirdly, it enhances biocontrol and microbial colonization. Bacillus megaterium, as a biocontrol bacterium, secretes antimicrobial peptides to effectively control common root rot diseases in saline-alkali land; Trichoderma echinosporum inhibits soil-borne pathogenic fungi through hyperparasitism, and its hyphal network expands the colonization space for bacterial communities, improving the migration ability of inoculants in compacted soil; Trichoderma, together with halophilic and potassium-solubilizing bacteria, forms a synergistic system of fungi and bacteria, further enhancing the stability and persistence of the microbial community under saline-alkali stress.

[0041] Regarding the stability of the microbial agent, this agent constructs a multi-layered stress-resistance system through staged fermentation and the application of electrochemical low-Hertz water throughout the entire process: the biofilm formed by Bacillus subtilis and the Trichoderma mycelial network jointly encapsulate the functional bacterial community, buffering the impact of salt and pH fluctuations; the metabolites of Haloxylon ammodendron enhance the integrity of the bacterial cell membrane; and the addition of salt-tolerant bacteria at the end of fermentation further consolidates the salt tolerance of the system, making the viable cell retention rate of the agent significantly better than that of conventionally processed products during storage and application.

[0042] The high efficiency and adaptability of the microbial agent stem from the precise matching of the sequential activation of microbial functions with soil problems: the first stage creates a low-salt microenvironment, clearing the way for the survival of subsequent functional microorganisms; the second stage, with the support of existing nitrogen sources, rapidly releases available phosphorus and exerts its fertility supply function through phosphorus-solubilizing bacteria; the third stage, with potassium-solubilizing bacteria and synergistic strains, further releases potassium and other nutrients fixed in the soil, while optimizing the rhizosphere microecology. This sequential design of "resistance first, function later" precisely addresses the core remediation challenges of saline-alkali land, namely "salt inhibition, alkali fixation, and low microbial activity," allowing the microbial agent to simultaneously achieve the compound remediation goals of soil desalination, nutrient activation, and crop growth promotion at relatively low application rates.

[0043] From the perspective of the formulation and fermentation strategy of the liquid compound microbial agent, the proportion of remediation bacteria (salt-tolerant bacteria) is the highest (3 parts), reflecting the saline-alkali land-specific strategy of prioritizing salt reduction; nitrogen-fixing bacteria are the second highest (2 parts), serving as the core of nutrient supply; growth-promoting bacteria (1.5 parts) enhance growth regulation; phosphorus-solubilizing, potassium-solubilizing, and biocontrol bacteria each account for 1 part, while synergistic bacteria account for only 0.5 parts, ensuring complete functionality while avoiding excessive competition between fungi and bacteria. This invention, through the design of a stress-resistant, nutrient-enhancing, and efficiency-enhancing microbial community structure and a three-stage dynamic fermentation process, forms a complete and adaptive saline-alkali land remediation microbial system. Combined with the full-process enhancement effect of electrochemical low-Hertz water on microbial community metabolism and stress resistance, the yield, functional activity, and field colonization rate of the microbial agent are significantly improved, providing an efficient, stable, and scalable microbial solution for saline-alkali land management.

[0044] In the preparation of the liquid compound microbial agent, during the first stage of fermentation to the middle of the logarithmic growth phase (12-16 hours), betaine, sodium citrate, and sodium chloride are added in a pulsed manner. At this stage, the metabolic activity of halophilic and nitrogen-fixing bacteria is highest, and the absorption efficiency of osmotic protectants and ion chelating agents is also optimal. Betaine, as a key compatible solute, can directly enhance the salt tolerance of the cells; sodium citrate, through chelation of Ca... 2+ / Mg 2+ To reduce ion toxicity; low concentrations of sodium chloride (0.3%) can induce the expression of salt-tolerant genes without inhibiting nitrogen fixation activity. All three are added in pulses with a strictly controlled order (NaCl is added 30 seconds in advance) to gradually establish the strain's salt tolerance and ensure the stability and activity of nitrogenase in a low-salt microenvironment.

[0045] When the first stage of fermentation is complete and the system pH stabilizes at 7.8±0.2, the second stage is initiated by adding calcium phytate, L-proline, and MnSO4. Calcium phytate, as a specific substrate for phosphate-solubilizing bacteria, is continuously fed in at a rate of 0.15 g / L·h to induce phytase secretion. L-proline enhances the salt stress resistance of growth-promoting bacteria and promotes IAA (indoleacetic acid) synthesis. MnSO4 activates the key enzyme in the biocontrol bacteria that produces antimicrobial peptides. The three are added sequentially: calcium phytate first, followed by L-proline and MnSO4 one hour later, to avoid nutrient competition and achieve simultaneous activation and synergistic effects of phosphate solubilization, growth promotion, and biocontrol.

[0046] After initiating the third stage and inoculating with potassium-solubilizing bacteria, potassium feldspar powder, humic acid, and ZnSO4 were added in batches. 200-mesh potassium feldspar powder (10 g / L) was added in two separate additions (5 hours apart) to prevent the system viscosity from exceeding 45 cP, which would affect dissolved oxygen efficiency. Humic acid improved the high pH environment and promoted Trichoderma spore germination; ZnSO4 activated the cellulase-producing genes in Trichoderma. Potassium feldspar powder was added first, followed by humic acid and ZnSO4 via atomization 30 minutes later, ensuring sufficient contact between the substrate and the bacterial cells. Immediately after inoculation with *Trichoderma echinosporum*, mechanical stirring (160-200 rpm) was started to raise the dissolved oxygen to 40-45%, meeting the strict aerobic requirements of *Trichoderma* while simultaneously promoting potassium feldspar decomposition and organic acid secretion.

[0047] The bio-fertilizer for saline-alkali soils provided by this invention utilizes the aforementioned liquid compound microbial agent suitable for saline-alkali soils. This agent possesses functions such as salt reduction, nitrogen fixation, phosphorus and potassium solubilization, growth promotion, and biocontrol synergistic effects, enabling the construction of a self-sustaining micro-ecosystem in high-salt and high-alkali soils. Therefore, the bio-fertilizer provided by this invention is highly adapted to the core pain points of saline-alkali soils, including high salinity, high pH, ​​nutrient fixation, and low microbial activity. It not only effectively reduces soil EC value and pH and activates fixed nutrients but also promotes crop growth and development under salt stress. Furthermore, this fermentation process has clearly defined process parameters and process control indicators, making it suitable for industrial scale-up and automated production. This ensures the stability and reproducibility of the microbial agent's effects in field applications, providing an efficient, controllable, and scalable microbial remediation solution for saline-alkali soil management. Compared with ordinary chemical fertilizers and conventional microbial fertilizers, the bio-fertilizer provided by this invention, relying on industrial fermentation technology and the synergistic advantages of multifunctional microbial communities, exhibits significant high efficiency and stability in practical applications: the application amount per unit area is reduced by 73.87-82.63% compared to urea, and it can achieve a 18.6-22.4% reduction in soil pH, a 31.2-35.8% decrease in sodium ion adsorption ratio (SAR), a 25.0% increase in organic matter content, and a 38.9% increase in available phosphorus content. Simultaneously, it improves crop root vitality by 65.5-70.8%, reduces disease incidence by 43.3-48.0%, and increases yield by 18.6-21.3%, achieving the synergistic goals of increased crop yield, improved quality, and improved soil health. This invention solves the problem of large application amounts caused by unstable efficacy after application in bio-agents and microbial fertilizers made from single strains or simple combinations of strains. Detailed Implementation

[0048] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading this description, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0049] Example 1: Preparation of a liquid compound microbial agent suitable for soil microecological remediation in saline-alkali land

[0050] The liquid compound microbial agent for the microecological remediation of saline-alkali soil described in this embodiment includes eight functional strains: salt-tolerant bacteria (Bacillus subtilis, Nocardiopsishalotolerans), nitrogen-fixing bacteria (Azotobacter chroococcum), phosphate-solubilizing bacteria (Pseudomonas fluorescens), potassium-solubilizing bacteria (Paenibacillus mucilaginosus), growth-promoting bacteria (Azospirillum brasilense), biocontrol bacteria (Pseudomonas putida), and synergistic bacteria (Trichoderma asperellum).

[0051] Eight bacterial strains were inoculated from preservation slant agar to specialized slant culture media. *Bacillus subtilis* was inoculated onto LB medium supplemented with 0.3% NaCl; *Haloxylon ammodendron* was inoculated onto ISP2 medium supplemented with 0.5% NaCl; nitrogen-fixing bacteria and growth-promoting bacteria were inoculated onto Asbestos medium; phosphate-solubilizing bacteria were inoculated onto KB medium; biocontrol bacteria were inoculated onto LB medium; potassium-solubilizing bacteria were inoculated onto potassium-solubilizing medium; and synergistic bacteria were inoculated onto PDA medium. All seven culture media were prepared using electrolytic low-Hertz water and sterilized at 121°C for 20 minutes. The cultures were then incubated at 30°C for 24-48 hours until the logarithmic growth phase (OD50). 600 When the concentration was ≥1.0, the samples were transferred to shake flasks for culture, and then transferred to seven primary seed tanks for culture.

[0052] When the bacterial count in the primary seed tank is ≥1×10 9 At CFU / mL, Bacillus subtilis was in the logarithmic growth phase (cultured for 8-12 hours). Bacillus subtilis, Haloxylon ammodendron, and Azotobacter chrysogenum were mixed in a 2:1:2 ratio to obtain premixed seed solution 1. Pseudomonas fluorescens, Azotobacter brasiliensis, and Bacillus megaterium seed solutions were mixed in a 2:3:2 volume ratio to obtain premixed seed solution 2. After incubation at 30°C for 30 minutes, the two premixed seed solutions were used for sequential fermentation.

[0053] When the number of Bacillus mucilaginosus and Bacillus subtilis bacteria in the primary seed tank is ≥1×10 9 At CFU / mL, the number of Trichoderma acicularis spores is ≥1×10⁻⁶. 7 When the spore count is 1 mL, it can be used for sequential fermentation.

[0054] The inoculation amounts of the premixed seed solution 1, premixed seed solution 2, Bacillus mucilaginosus seed solution, Trichoderma echinosporum seed solution, and Bacillus subtilis seed solution (culture in the early stage of the stable period (18-24h)) are 11.8%-12.2%, 6.8%-7.2%, 2.8%-3.2%, 2.8%-3.2%, and 2.8%-3.2% of the fermentation volume, respectively. The inoculation amount of electrochemical low-hertz water is 71.4%-72.6% of the fermentation volume. The electrochemical low-hertz water has a molecular cluster of ≤6 molecules and a pH of 7.0-7.5.

[0055] The subsequent three-stage sequential culture was carried out in the main fermenter:

[0056] S1: Propagation of salt-tolerant and nitrogen-fixing bacteria: Electrolyzed low-Hertz water is added to the sterilized main fermenter, along with a pre-mixed mixture of Bacillus subtilis, Haloxylon ammodendron, and Azotobacter chrysogenum. The fermentation conditions are adjusted to the first-order fermentation conditions: pH = 7.5 ± 0.3, dissolved oxygen 25-35%, temperature 32℃, humidity at the top of the fermenter 90-92%, and fermentation time 34-38 hours. During the S1 stage, when fermentation reaches the middle of the logarithmic growth phase, the growth factors betaine, sodium citrate, and sodium chloride are added. Betaine, sodium citrate, and sodium chloride are added in batches using a pulse addition method: the final concentration of betaine is 0.04% (w / v), the final concentration of sodium citrate is 0.15% (w / v), and the final concentration of sodium chloride is 0.3% (w / v); these are added in 3 batches, 3 hours apart, with each addition completed within 10 minutes; the order of addition is: first add sodium chloride, then simultaneously add betaine and sodium citrate 30 seconds later.

[0057] S2: Introduction of phosphate-solubilizing bacteria, growth-promoting bacteria, and biocontrol bacteria: After the S1 stage, adjust to the second-sequence fermentation conditions, and add pre-mixed seed cultures of *Pseudomonas fluorescens*, *Azotobacter brasiliensis*, and *Bacillus megaterium* to the main fermenter. The second-sequence fermentation conditions are: pH = 7.8 ± 0.3, dissolved oxygen 30-40%, temperature 32℃, humidity at the top of the fermenter 91-93%, and fermentation time 16-20 hours. After the S1 stage fermentation is completed and the system pH stabilizes at 7.8 ± 0.2, add growth factors calcium phytate, L-proline, and MnSO4. After inoculating potassium-solubilizing bacteria in the S3 stage, add growth factors potassium feldspar powder, humic acid, and ZnSO4. Calcium phytate was added continuously at a flow rate of 0.15 g / L·h to a final concentration of 1.8 g / L; L-proline and MnSO4 were added simultaneously at a flow rate of 0.002 g / L·h to final concentrations of 0.03% (w / v) and 0.005% (w / v), respectively; one hour after the calcium phytate addition was started, the addition of L-proline and MnSO4 was then started.

[0058] S3: Initiate the synergistic metabolism of potassium-solubilizing bacteria and Trichoderma, and supplement with salt-tolerant bacteria: After the fermentation of S2 stage, after Bacillus mucilaginosus seed liquid is introduced into the main fermenter and fermented for 12 hours, the fermentation parameters are monitored (pH should drop to 6.5-7.0, dissolved oxygen >50%). Bacillus subtilis seed liquid (3% inoculum) is introduced, and mechanical stirring (200-300 rpm) is started immediately. Dissolved oxygen is maintained at 60%, temperature at 28℃. After fermentation for 12 hours, Trichoderma echinosporum seed liquid (3% inoculum) is introduced to enter the fungal fermentation stage, and the fermentation conditions are adjusted to the third time sequence: pH=7.5±0.3, dissolved oxygen 40-450%, temperature at 32℃, humidity at the top of the fermenter 85-88%, fermentation for 40-44 hours; After inoculating potassium-solubilizing bacteria in S3 stage, growth factors potassium feldspar powder, humic acid and ZnSO4 are added. Potassium feldspar powder, humic acid, and ZnSO4 were added in batches and via atomization: the total concentration of potassium feldspar powder with a particle size of 200 mesh was 10 g / L, added in two batches of 5 g / L each, with an interval of 5 hours; the final concentrations of humic acid and ZnSO4 were 0.2% (w / v) and 0.003% (w / v), respectively, and were added via simultaneous atomization with droplet diameters of 10-15 μm, starting simultaneously with the second addition of potassium feldspar powder; the potassium feldspar powder was added first, and the atomization of humic acid and ZnSO4 began 30 minutes later.

[0059] When the number of viable bacteria in the main fermenter is ≥2×10 10 Fermentation ends when CFU / mL, yielding the liquid compound microbial agent suitable for soil microecological restoration in saline-alkali land.

[0060] In some embodiments of the present invention, the fermentation equipment used in the above fermentation process includes:

[0061] The main fermentation tank includes a three-layer inclined blade agitator, jacketed heating / cooling, three humidity sensors, a saturated humidifier, a refrigerated dehumidifier, a hot air circulation device, a condensate return and discharge system, an automated control system, and a functional detection module.

[0062] In some embodiments of the present invention, the main fermentation tank has a volume of 3000L, and the humidity sensors are respectively placed on the top / middle / bottom of the tank, spaced 15cm apart; the saturated humidifier has an adjustable water temperature of 30-32℃ and a processing capacity of 6m³. 3 / h; the refrigerated dehumidifier can cool down to 15℃, and the humidity after dehumidification is ≤80%; the hot air circulation device has an air temperature of 30℃±1℃ and an air speed of 0.5-1.2m / s, which can be adjusted as needed; the condensate return and discharge system is equipped with a 0.22μm sterile filter membrane; the automatic control system includes PLC linkage functions for humidity, temperature, dissolved oxygen, and stirring speed, with a response time of <10s, and can preset parameter curves for dryland farming; the functional detection module monitors the effective phosphorus / potassium concentration online and detects viable bacteria count, proline, ferrophilic acid, and nitrogenase activity offline.

[0063] In some embodiments of the present invention, the humidity of the space above the fermentation tank is regulated by the humidity sensor, the saturated humidifier, and the refrigerated dehumidifier.

[0064] Example 2: Preparation of bio-fertilizer for soil micro-ecological restoration in saline-alkali land

[0065] The bio-fertilizer described in this embodiment is prepared using the liquid compound microbial agent for soil micro-ecological restoration suitable for saline-alkali land described in Example 1.

[0066] In a specific embodiment of the present invention, the raw materials of the bio-fertilizer include: liquid compound microbial agent, electrochemical low-hertz water, and solid components: lignite powder, wheat bran, and soybean meal; the total amount of the solid components is 100%, and the proportions of the three components are: lignite powder 70-85%, wheat bran 8-15%, and soybean meal 8-20%; the particle size of the lignite powder is 80-100 mesh, and the initial moisture content is 10-15%; the initial moisture content of the wheat bran and soybean meal is 8-12% each. After the above solid components are mixed evenly, electrochemical low-hertz water is added and stirred thoroughly. At the same time, the liquid compound microbial agent is added simultaneously during the stirring process, and the amount added is calculated as 0.8-10% of the total amount of solid components, so that the total moisture content of the mixture reaches 50-60%.

[0067] The method for preparing the bio-fertilizer includes:

[0068] A1: The liquid compound microbial agent suitable for saline-alkali land, lignite powder, wheat bran, soybean meal, and electrolytic low-hertz water are added to the intelligent mixing system. After uniform mixing, the mixture is conveyed to the intelligent solid-state fermentation system production line through the independently invented intelligent material distribution system (patent authorization announcement number CN 116553963B) for the first solid-state fermentation. The intelligent fermentation system regulates the temperature during the fermentation process through temperature sensors and linkage with intermittent micro-burst oxygen frequency: the initial stage temperature is 42-45℃, maintained for 22-24 hours; the peak stage temperature is 60-65℃, maintained for 46-48 hours; and the final stage temperature is 40-45℃, maintained for 22-24 hours. When the temperature remains constant for 24 consecutive hours, the material moisture content is ≤40%, the material color is dark brown, there is no odor, and the material is loose to the touch without sticking, the fermentation of stage A1 ends.

[0069] A2: The material obtained from the first fermentation in A1 is rearranged into the aging and fermentation production line through an intelligent material distribution system for the second aging and fermentation: The material is naturally piled up, and combined with intelligent monitoring, the residual heat and residual microorganisms of the material are used to complete the deep decomposition, which lasts for 10-12 days. When the material naturally cools down to a moisture content of 15-20%, the color is uniformly dark brown, the texture is loose, it has an earthy smell without other odors, the pH is stable at 6.5-7.5, and the number of beneficial live bacteria is ≥200 million / g, the second fermentation is completed, and the bio-fertilizer suitable for saline-alkali land is obtained.

[0070] Experiment Example 1: Effect of Electrochemical Low-Hertz Water on the Number of Viable Cells at Different Stages of Fermentation

[0071] Experimental group: Liquid compound microbial agent suitable for saline-alkali land prepared according to the method of Example 1.

[0072] Control group: The electrochemical low-hertz water in Example 1 was replaced with sterilized water, and all other operations were the same as in Example 1.

[0073] The number of viable bacteria in the experimental and control groups at each fermentation stage was measured. Statistical analysis was performed using ANOVA (α=0.05), with at least three replicates per group. The results are shown in Table 1. At the end of the first time step, the number of viable bacteria in the experimental group increased by 56% compared to the control group; at the end of the second time step, the number of viable bacteria in the experimental group increased by 50% compared to the control group; and at the end of the third time step, the number of viable bacteria in the experimental group increased by 42% compared to the control group. This indicates that electrochemical low-Hertz water can effectively improve fermentation efficiency.

[0074] Table 1. Viable bacterial count

[0075]

[0076] Experimental Example 2: The Effect of Bio-fertilizer Applicable to Saline-Alkali Land

[0077] 2.1 Wheat Experiment

[0078] (1) Test location:

[0079] Hengshui City, Hebei Province, saline-alkali soil, pH: 8.6±0.2, EC value: 4.2±0.3 (dS / m), SAR value: 18.5±1.2 (mmol / m). 1 / 2 ·L -1 / 2 Available nitrogen: 78.2±3.1 mg / kg, available phosphorus: 6.8±0.5 mg / kg, available potassium: 92±4.8 mg / kg, Na in the 0-20cm soil layer + Content: 0.35±0.03%.

[0080] (2) Test fertilizers and fertilization methods:

[0081] Processing Group 1:

[0082] Fertilizer: Bio-fertilizer 1 was obtained according to the preparation method of Example 2, with the amount of liquid compound microbial agent added being 10%.

[0083] Application methods: Base fertilizer: Shiluyuan commercial organic fertilizer (900 kg / mu); bio-fertilizer 1: seed dressing (1.6 kg / mu); irrigation during the greening stage (8 kg / mu); spraying during the jointing stage (10 kg / mu).

[0084] Processing Group 2:

[0085] Fertilizer: Replace the small molecule cluster water in the preparation method of Example 2 with ordinary sterilized water, and perform the other operations exactly the same as in Example 2 to obtain bio-fertilizer 2.

[0086] Application method: Same as treatment group 1.

[0087] Control group 1:

[0088] Fertilizer: The liquid compound bacterial agent in the preparation method of Example 2 is replaced with a single brown azotocin agent, and the other operations are exactly the same as in Example 2, to obtain a single nitrogen-fixing fertilizer.

[0089] Application method: Same as treatment group 1.

[0090] Control group 2:

[0091] Fertilizers: Sinochem Compound Fertilizer (18-22-8), Luyuan Commercial Organic Fertilizer, Sinochem Urea (≥46.4%), Jiheng Group Urea.

[0092] Application method: Apply Sinochem compound fertilizer (18-22-8) (50 kg / mu) and Luyuan commercial organic fertilizer (900 kg / mu) as base fertilizer; apply Sinochem urea (≥46.4%) (15 kg / mu) during the greening stage; apply Jiheng Group urea (10 kg / mu) during the jointing stage.

[0093] (3) Experimental design:

[0094] The wheat variety planted was "Jimai 418" (which has strong salt tolerance and is the main variety planted in saline-alkali areas). The randomized block design was used (2 mu per group, 3 replicates). Basal fertilizer was applied to each group one week before sowing, and irrigation was carried out in "small amounts and multiple times" (to avoid salt stratification). Weeding was carried out in a consistent manner. Only control group 2 was sprayed with jinggangmycin (50g / mu) due to sheath blight, while the other groups were not treated.

[0095] (4) Experimental results:

[0096] The experimental results are shown in Table 2 below. Compared with treatment group 2 and control group 1, treatment group 1 showed significantly higher effective tiller number, grain filling rate, thousand-grain weight, yield per unit area, pH reduction, EC reduction, SAR reduction, and available phosphorus content in the soil, and significantly lower sheath blight incidence index. This indicates that bio-fertilizer 1, i.e., the bio-fertilizer suitable for saline-alkali land provided by this invention, is significantly more effective than bio-fertilizer 2 in treatment group 2 and nitrogen-fixing fertilizer in control group 1 under the same application method and dosage, and can effectively promote wheat growth, increase yield, and improve soil. Compared with control group 2, treatment group 1 showed significantly higher effective tiller number, grain filling rate, thousand-grain weight, yield per unit area, pH reduction, EC reduction, SAR reduction, and available phosphorus content in the soil, and significantly lower sheath blight incidence index. This indicates that the dosage of bio-fertilizer 1, i.e., the bio-fertilizer provided by this invention for saline-alkali land, is much lower than that of ordinary commercial fertilizers and organic fertilizers, has high applicability to saline-alkali land, and its growth-promoting and soil-remediation effects are significantly better than those of ordinary commercial fertilizers and organic fertilizers.

[0097] Table 2 Results of wheat planting experiment

[0098]

[0099] 2.2 Rice Experiment 1

[0100] (1) Test location:

[0101] Jilin Province, soda-saline soil, pH: 8.8±0.2, EC value (dS / m): 3.8±0.3, SAR value: 16.8±1 (mmol / m). 1 / 2 ·L -1 / 2 Available phosphorus: 7.2±0.4 (mg / kg), available potassium: 152±4.5 (mg / kg).

[0102] (2) Test fertilizers and fertilization methods:

[0103] Processing Group 1:

[0104] Fertilizer: The amount of liquid compound microbial agent added to the bio-fertilizer 1 prepared according to the preparation method of Example 2 is 6%.

[0105] Application method: Apply Luyuan commercial organic fertilizer (550 kg / mu) as base fertilizer; mix seed with bio-fertilizer 1 (1.5 kg / mu); broadcast bio-fertilizer 1 (6 kg / mu) during tillering stage; spray bio-fertilizer 1 (9 kg / mu) during booting stage; spray bio-fertilizer 1 (1 kg / mu) at the early grain-filling stage. Precautions: Spray on a sunny evening, focusing on the underside of the leaves. If it rains within 6 hours after spraying, re-spray.

[0106] Processing Group 2:

[0107] Fertilizer: Replace the small molecule cluster water in the preparation method of Example 2 with ordinary sterilized water, and perform the other operations exactly the same as in Example 2 to obtain bio-fertilizer 2.

[0108] Application method: Same as treatment group 1.

[0109] Control group 1:

[0110] Fertilizer: The liquid compound bacterial agent in the preparation method of Example 2 is replaced with a single brown azotocin agent, and the other operations are exactly the same as in Example 2, to obtain a single nitrogen-fixing fertilizer.

[0111] Application method: Same as treatment group 1.

[0112] Control group 2:

[0113] Fertilizers: Sinochem Fertilizer (18-22-8), Sinochem Urea (≥46.4%), Sinochem Potassium Chloride (containing 60% K2O), Stanley Potassium Dihydrogen Phosphate (P2O5 52%, K2O 34%).

[0114] Application method: Before transplanting, evenly spread Sinochem (18-22-8) (50 kg / mu) and Luyuan commercial organic fertilizer (550 kg / mu), and rotary till into the soil; Transplanting period: Spread Sinochem urea (≥46.4%) (8 kg / mu); Tillering period: Spread Sinochem urea (≥46.4%) (12 kg / mu) + Sinochem potassium chloride (containing 60% K2O) (8 kg / mu); Heading period: Spread Sinochem urea (containing 46% nitrogen) (5 kg / mu) + Sinochem potassium chloride (containing 60% K2O) (10 kg / mu); Early grain filling stage: Spray Sinochem urea (≥46.4%) (2 kg / mu) + Stanley potassium dihydrogen phosphate (P2O 552%, K2O 34%) (150 g / mu).

[0115] (3) Experimental design:

[0116] The rice variety “Jijing 816” (tolerant to sodium chloride and alkalinity, a major variety cultivated in Northeast China) was planted using a randomized block design, with each group having a planting area of ​​1.5 mu (approximately 0.16 acres) and three replicates. Field management was consistent across all groups.

[0117] (4) Test results

[0118] The experimental results are shown in Table 3 below: Compared with treatment group 2 and control group 1, treatment group 1 showed significantly improved root activity, stem thickness, chlorophyll SPAD value, yield per unit area, pH reduction, EC value reduction, and SAR value reduction, and the tillering peak period was earlier. This indicates that bio-fertilizer 1, i.e., the bio-fertilizer suitable for saline-alkali land provided by this invention, is significantly more effective than bio-fertilizer 2 in treatment group 2 and nitrogen-fixing fertilizer in control group 1 under the same application method and dosage, and can effectively promote rice growth and improve soil properties. Compared with control group 2, treatment group 1 showed significantly improved root activity, stem thickness, chlorophyll SPAD value, yield per unit area, pH reduction, EC value reduction, and SAR value reduction, and the tillering peak period was earlier. This indicates that the dosage of bio-fertilizer 1, i.e., the bio-fertilizer suitable for saline-alkali land provided by this invention, is much lower than that of ordinary commercial fertilizers and organic fertilizers, and its ability to promote rice growth, increase yield, and adapt to saline-alkali land is significantly better than that of ordinary commercial fertilizers and organic fertilizers.

[0119] Table 3 Results of Rice Experiment 1

[0120]

[0121] 2.3 Rice Experiment Two

[0122] (1) Experimental location: Tianjin, coastal saline soil, pH: 8.7±0.2, EC value: 4.0±0.2 (dS / m), SAR value: 17.8±1.1 (mmol) 1 / 2 ·L -1 / 2 Available phosphorus: 5.9 ± 0.4 (mg / kg). Available potassium: 88 ± 4.5 (mg / kg).

[0123] (2) Test fertilizers and fertilization methods:

[0124] Except for the addition amount of liquid compound microbial agent, which was 0.8% in Rice Experiment 1, the two experiments were completely identical.

[0125] (3) Experimental design:

[0126] The rice variety planted was "Binyan 14". A randomized block design was used: 1.5 mu per group, with 3 replicates and consistent field management.

[0127] (4) Test results

[0128] The experimental results are shown in Table 4 below: Compared with treatment group 2 and control group 1, treatment group 1 showed significantly improved root activity, stem thickness, chlorophyll SPAD value, yield per unit area, pH reduction, EC value reduction, and SAR value reduction, and the tillering peak period was earlier. This indicates that bio-fertilizer 1, i.e., the bio-fertilizer suitable for saline-alkali land provided by this invention, is significantly more effective than bio-fertilizer 2 in treatment group 2 and nitrogen-fixing fertilizer in control group 1 under the same application method and dosage, and can effectively promote rice growth and improve soil properties. Compared with control group 2, treatment group 1 showed significantly improved root activity, stem thickness, chlorophyll SPAD value, yield per unit area, pH reduction, EC value reduction, and SAR value reduction, and the tillering peak period was earlier. This indicates that the dosage of bio-fertilizer 1, i.e., the bio-fertilizer suitable for saline-alkali land provided by this invention, is much lower than that of ordinary commercial fertilizers and organic fertilizers, and its ability to promote rice growth, increase yield, and adapt to saline-alkali land is significantly better than that of ordinary commercial fertilizers and organic fertilizers.

[0129] Table 4 Results of Rice Experiment 2

[0130]

[0131] 3. Summary of Results Comparison

[0132] 3.1 Comparison of the effects of bio-fertilizers produced using liquid compound microbial agents and single nitrogen-fixing microbial agents

[0133] This invention utilizes a combination of eight functional strains of salt-tolerant bacteria: Bacillus subtilis, Haloxylon ammodendron, Azotobacter chrysogenum (nitrogen fixation), Pseudomonas fluorescens (phosphate solubilization), Bacillus mucilaginosus (potassium solubilization), Azotobacter brasiliensis (growth promotion), Bacillus megaterium (biocontrol), and Trichoderma echinosporum (antagonism) to prepare a bio-fertilizer (treatment group 1). Through the synergistic effects of salt tolerance, nitrogen fixation, phosphorus solubilization, potassium solubilization, growth promotion, and biocontrol, the growth promotion effect is significantly better than that of a single nitrogen-fixing bacteria agent (control group 1).

[0134] In the wheat experiment: compared with the control group 1, treatment group 1 showed an increase of 29.6% in effective tiller number, 33.3% in grain filling rate, 11.5% in thousand-grain weight, 18.6% in yield per unit area, 166.7% in pH reduction, 150.0% in EC reduction, 148.0% in SAR reduction, 73.6% in available phosphorus content in soil, and 58.3% in sheath blight incidence index (Table 2).

[0135] In the first rice experiment: compared with the control group 1, treatment group 1 showed an increase of 48.6% in root activity, 14.5% in stem thickness, 21.4% in chlorophyll SPAD value, 15.2% in yield per unit area, 125.0% in pH reduction, 180.0% in EC value reduction, 152.2% in SAR value reduction, and 4 days earlier in the peak tillering period (Table 3).

[0136] In the second rice experiment: root activity increased by 48.9%, stem thickness increased by 14.8%, chlorophyll SPAD value increased by 22.0%, yield increased by 14.3%, pH decrease increased by 166.7%, EC value decrease increased by 160.0%, SAR value decrease increased by 150.0%, and the peak tillering period was advanced by 4 days (Table 4).

[0137] 3.2 Comparison of the effects of electrochemical low-hertz water and ordinary sterilized water

[0138] This invention uses electrochemical low-hertz water with a half-width at half-maximum (WHM) of ≤100Hz to prepare bio-fertilizer 1 (treatment group 1). By improving the permeability of microbial cell membranes and enhancing the adhesion and diffusion of bacterial agents, it is significantly superior to bio-fertilizer 2 (treatment group 2) made with ordinary water.

[0139] In the wheat experiment: compared with treatment group 2, treatment group 1 showed an increase of 12.9% in effective tiller number, 14.3% in grain filling rate, 6.6% in thousand-grain weight, 8.5% in yield per unit area, 60.0% in pH reduction, 50.0% in EC reduction, 51.2% in SAR reduction, 27.6% in available phosphorus in soil, and 33.3% in sheath blight incidence index (Table 2).

[0140] In the first rice experiment: root activity increased by 18.5%, stem thickness increased by 6.0%, chlorophyll SPAD value increased by 8.5%, yield per unit area increased by 6.5%, pH decrease increased by 50.0%, EC value decrease increased by 55.6%, SAR value decrease increased by 48.7%, and the peak tillering period was advanced by 2 days (Table 3).

[0141] In the second rice experiment: root activity increased by 17.5%, stem thickness increased by 6.1%, chlorophyll SPAD value increased by 8.7%, yield per unit area increased by 6.7%, pH decrease increased by 60.0%, EC value decrease increased by 44.4%, SAR value decrease increased by 48.6%, and the tillering peak period was advanced by 2 days (Table 4).

[0142] 3.4 Summary

[0143] This invention quantified and verified the technical effects of increasing yield, improving quality, reducing disease, and improving soil through planting trials of two crops in three regions: Treatment group 1, specifically the bio-fertilizer 1 provided by this invention suitable for saline-alkali land, showed significant growth-promoting effects in three growth-promoting trials on wheat and rice, not only increasing yield and quality but also significantly improving soil (Table 5). This indicates that the bio-fertilizer is suitable for grain crops on saline-alkali land, not only promoting growth and increasing yield but also effectively improving soil properties and enhancing root vitality.

[0144] Table 5. Comparison of effects between treatment group 1 and control group 2

[0145]

[0146] In summary, the bio-fertilizer for saline-alkali land provided by this invention achieves functional complementarity and rhizosphere ecological regulation through eight functional bacteria, coupled with electrochemical low-Hertz water enhancement and solid-state fermentation technology, thereby increasing the yield and quality of wheat and rice in saline-alkali land, reducing the salt content in the soil, and increasing the organic matter content in the soil.

Claims

1. A microecological restoration liquid compound microbial agent suitable for saline-alkali land, characterized in that, The following eight functional strains were prepared using electrochemical low-hertz water, i.e., small molecule cluster water, through a three-stage sequential inoculation and fermentation process: Salt-tolerant bacteria (Bacillus subtilis, Nocardiopsis halotolerans), nitrogen-fixing bacteria (Azotobacter chroococcum), phosphate-solubilizing bacteria (Pseudomonas fluorescens), potassium-solubilizing bacteria (Paenibacillus mucilaginosus), growth-promoting bacteria (Azospirillum brasilense), biocontrol bacteria (Pseudomonas putida), and synergistic bacteria (Trichoderma asperellum). S1: Propagation of salt-tolerant and nitrogen-fixing bacteria: Add electrolytic low-hertz water to the sterilized main fermenter, and add pre-mixed Bacillus subtilis, Haloxylon ammodendron, and Azotobacter spp. Adjust the fermentation conditions to the first-order fermentation conditions: pH=7.5±0.3, dissolved oxygen 25-35%, temperature 32℃, humidity of the top space of the fermenter 90-92%, fermentation time 34-38 hours; S2: Introduction of phosphate-solubilizing bacteria, growth-promoting bacteria, and biocontrol bacteria: After the S1 stage, the conditions are adjusted to the second fermentation sequence, and pre-mixed seed liquids of *Pseudomonas fluorescens*, *Azotobacter brasiliensis*, and *Bacillus megaterium* are added to the main fermenter. The second fermentation sequence conditions are: pH=7.8±0.3, dissolved oxygen 30-40%, temperature 32℃, humidity of the top space of the fermenter 91-93%, and fermentation time 16-20 hours. S3: Initiate the synergistic metabolism of potassium-solubilizing bacteria and Trichoderma, and supplement with salt-tolerant bacteria: After the fermentation of the S2 stage, Bacillus mucilaginosus seed liquid is introduced into the main fermenter and fermented for 12 hours. After fermentation, the parameters in the fermenter are monitored. When the pH drops to 6.5-7.0 and the dissolved oxygen is >50%, Bacillus subtilis seed liquid is introduced. Mechanical stirring is started immediately, with the speed set to 200-300 rpm, maintaining dissolved oxygen at 60% and temperature at 28℃. After fermentation for 12 hours, Trichoderma echinococcosis seed liquid is introduced, and the fungal fermentation stage is entered. The fermentation conditions are adjusted to the third time sequence: pH=7.5±0.3, dissolved oxygen 40-45%, temperature 32℃, humidity of the top space of the fermenter 85-88%, and fermentation time 40-44 hours. When the number of viable bacteria in the main fermenter is ≥2×10 10 Fermentation ends when CFU / mL is reached.

2. The microecological restoration liquid compound microbial agent suitable for saline-alkali land according to claim 1, characterized in that, The ratio of the restorative bacteria to the ratio of salt-tolerant bacteria to nitrogen-fixing bacteria to phosphorus-solubilizing bacteria to potassium-solubilizing bacteria to growth-promoting bacteria to biocontrol bacteria to synergistic bacteria is 3:2:1:1:1.5:1:0.5, and the internal ratio of the restorative salt-tolerant bacteria is Bacillus subtilis to Haloxylon ammodendron = 2:

1.

3. The microecological restoration liquid compound microbial agent suitable for saline-alkali land according to claim 2, characterized in that, The seed culture preparation method is as follows: eight functional bacterial strains are inoculated from the preservation slant into a special slant culture medium and cultured at 30℃ for 24-48 hours until the logarithmic phase, i.e., OD. 600 When the value is ≥1.0, the samples were transferred to shake flasks for culture, and then transferred to eight primary seed tanks for independent culture. The method for preparing the premixed seed solution is as follows: when the bacterial count in the primary seed tank is ≥1×10⁻⁶. 9 At CFU / mL, Bacillus subtilis, Haloxylon ammodendron, and Azotobacter chrysogenum, cultured for 8-12 h until the logarithmic growth phase, were mixed in a 2:1:2 ratio to obtain premixed seed solution 1. Pseudomonas fluorescens, Azotobacter brasiliensis, and Bacillus megaterium seed solutions were mixed in a 2:3:2 volume ratio to obtain premixed seed solution 2. After incubation at 30℃ for 30 minutes, the two premixed seed solutions were used for sequential fermentation. When the number of Bacillus mucilaginosus and Bacillus subtilis bacteria in the primary seed tank is ≥1×10 9 At CFU / mL, the number of Trichoderma acicularis spores is ≥1×10⁻⁶. 7 At a concentration of spores / mL, it can be used for sequential fermentation; The *Bacillus subtilis* was cultured on LB medium supplemented with 0.3% NaCl, the *Haloxylon ammodendron* on ISP2 medium supplemented with 0.5% NaCl, nitrogen-fixing bacteria and growth-promoting bacteria on Asbestos medium, phosphate-solubilizing bacteria on KB medium, biocontrol bacteria on LB medium, potassium-solubilizing bacteria on potassium-solubilizing medium, and synergistic bacteria on PDA medium; all seven media were prepared using electrolytic low-Hertz water and sterilized at 121°C for 20 minutes. The inoculation amounts of the premixed seed solution 1, premixed seed solution 2, Bacillus mucilaginosus seed solution, Trichoderma echinosporum seed solution, and Bacillus subtilis seed solution cultured for 8-12 h to the logarithmic growth phase are 11.8%-12.2%, 6.8%-7.2%, 2.8%-3.2%, 2.8%-3.2%, and 2.8%-3.2% of the fermentation volume, respectively. The inoculation amount of electrochemical low-hertz water is 71.4%-72.6% of the fermentation volume. The electrochemical low-hertz water has a molecular cluster of ≤6 molecules and a pH of 7.0-7.

5.

4. The microecological restoration liquid compound microbial agent suitable for saline-alkali land according to claim 3, characterized in that, During the S1 stage, when fermentation reaches the middle of the logarithmic growth phase, the growth factors betaine, sodium citrate, and sodium chloride are added. After the S1 stage fermentation ends and the system pH stabilizes at 7.8±0.2, the growth factors calcium phytate, L-proline, and MnSO4 are added. After inoculation with potassium-solubilizing bacteria in the S3 stage, the growth factors potassium feldspar powder, humic acid, and ZnSO4 are added.

5. The microecological restoration liquid compound microbial agent suitable for saline-alkali land according to claim 4, characterized in that, In stage S1, betaine, sodium citrate, and sodium chloride were added in batches using a pulse addition method: the final concentration of betaine was 0.04%, the final concentration of sodium citrate was 0.15%, and the final concentration of sodium chloride was 0.3%; the addition was done in 3 batches, with an interval of 3 hours between each addition, and each addition was completed within 10 minutes; the addition order was: sodium chloride was added first, followed by betaine and sodium citrate 30 seconds later. In stage S2, calcium phytate was added continuously at a flow rate of 0.15 g / L·h to a final concentration of 1.8 g / L; L-proline and MnSO4 were added simultaneously at a flow rate of 0.002 g / L·h to final concentrations of 0.03% and 0.005%, respectively; one hour after the calcium phytate addition was started, the addition of L-proline and MnSO4 was then initiated. In stage S3, potassium feldspar powder, humic acid, and ZnSO4 were added in batches and via atomization: the potassium feldspar powder had a particle size of 200 mesh and a total concentration of 10 g / L, added in two batches of 5 g / L each, with an interval of 5 hours; the final concentrations of humic acid and ZnSO4 were 0.2% and 0.003%, respectively, added via simultaneous atomization with droplet diameters of 10-15 μm, starting simultaneously with the second addition of potassium feldspar powder; and the potassium feldspar powder was added first, followed by the atomization addition of humic acid and ZnSO4 30 minutes later.

6. A microecological restoration bio-fertilizer suitable for saline-alkali land, characterized in that, It is prepared using the liquid compound microbial agent suitable for soil microecological remediation in saline-alkali land as described in any one of claims 1-5.

7. The microecological restoration bio-fertilizer for saline-alkali land according to claim 6, characterized in that, Preparation methods include: A1: The liquid composite microbial agent, electrochemical low-hertz water, and solid components (lignite powder, wheat bran, and soybean meal) described in claim 1 are added to a mixing system. After the mixture is evenly mixed, it is conveyed to the solid-state fermentation system production line through an intelligent material distribution system for the first solid-state fermentation. The solid-state fermentation system automatically adjusts the temperature during the fermentation process through a temperature sensor linked to the intermittent micro-burst oxygen frequency: the initial stage temperature is 42-45℃, maintained for 22-24 hours; the peak stage temperature is 60-65℃, maintained for 46-48 hours; and the final stage temperature is 40-45℃, maintained for 22-24 hours. A2: The material obtained from the first solid-state fermentation in A1 is rearranged into the aging and fermentation production line through an intelligent material distribution system for a second aging and fermentation: the naturally piled material is deeply decomposed using the residual heat and residual microorganisms. After 10-12 days, the fermentation ends, and the bio-fertilizer suitable for saline-alkali land is obtained.

8. The microecological restoration bio-fertilizer for saline-alkali land according to claim 7, characterized in that, In component A1, the total amount of solid components—lignite powder, wheat bran, and soybean meal—is 100%, with the following proportions: lignite powder 70-85%, wheat bran 8-15%, and soybean meal 8-20%. The lignite powder has a particle size of 80-100 mesh and an initial moisture content of 10-15%. The initial moisture content of the wheat bran and soybean meal is 8-12%. After the solid components are mixed evenly, electrolytic low-hertz water is added and stirred thoroughly. Simultaneously, a liquid compound microbial agent is added during the stirring process, at a rate of 0.8-10% of the total solid components, so that the total moisture content of the mixture reaches 50-60%.

9. The microecological restoration bio-fertilizer for saline-alkali land according to claim 8, characterized in that, The completion of the first fermentation stage A1 is indicated by the following: the temperature is maintained at the final stage temperature for 24 consecutive hours, the material moisture content is ≤40%, the material color is dark brown, there is no odor, it is loose when squeezed, and there is no stickiness.

10. The microecological restoration bio-fertilizer for saline-alkali land according to claim 9, characterized in that, The second fermentation in stage A2 is marked as follows: when the material naturally cools down to a moisture content of 15-29%, has a uniform dark brown color, a loose texture, an earthy smell without other odors, a pH of 6.5-7.5, and a beneficial live bacteria count ≥200 million / g, the second fermentation is complete.

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