Preparation method and application of high-activity chalcoatrum bacteria CML2 solid microbial agent

Abstract

The application discloses a preparation method and application of high-activity Achromobacter piechaudii CML2 solid microbial agent. The CML2 is subjected to high-density fermentation in a culture medium with sugarcane molasses as a carbon source, wet bacterial blocks are collected, mixed with a composite protective agent composed of 150 g / L of skimmed milk powder, 100 g / L of trehalose and 5 g / L of sodium glutamate and free of glycerol at a ratio of 1 g:1.5 mL, balanced at 37 DEG C, rapidly frozen in liquid nitrogen and vacuum freeze-dried to obtain loose porous bacterial powder with a water content of less than 5%. The freeze-drying survival rate of the preferred formula is 82.4%, the bacterial powder can be reconstituted within 30 s; after being stored in the dark at 4 DEG C and 25 DEG C for 180 days, the effective viable cell counts are not less than 3.8x10 9 CFU / g and 2.2x10 8 CFU / g, respectively. In field tests, seed soaking and application make the cadmium content of brown rice reduce by 46.2% and the thousand-grain weight increase by 10.8%.

Description

Technical Field

[0001] This invention belongs to the field of microbial preparations, specifically relating to a method for preparing and applying a highly active copper-containing bacillus CML2 solid microbial agent. Background Technology

[0002] With the rapid advancement of industrialization and urbanization, heavy metal pollution in farmland soil has become increasingly severe. Cadmium (Cd) pollution, in particular, has become a major threat to my country's food security and human health due to its high biotoxicity, ease of absorption by crops, and accumulation in the food chain. Rice, as a major food crop in my country, has a strong capacity for cadmium accumulation. Long-term consumption of rice with excessive cadmium (commonly known as "cadmium rice") can lead to serious health problems such as kidney damage and osteoporosis. The national food safety standard GB 2762-2022 stipulates that the cadmium limit for brown rice is 0.2 mg / kg. Microbial remediation technology has received widespread attention due to its low cost and minimal disturbance to soil structure.

[0003] Cupriavidus metallidurans, a type of microorganism with extremely high tolerance to heavy metals, has shown great potential in remediating polluted environments. CN117229951A discloses a cadmium-tolerant endophytic metal-tolerant copper-tolerant bacterium in rice and its application. This strain, CML2, exhibits extremely high tolerance (2400 mg / L) to cadmium, demonstrating strong cadmium resistance and highly efficient cadmium ion removal capabilities. It can significantly reduce the concentration of cadmium ions in the soil and mitigate its toxicity to organisms. Therefore, this strain can effectively achieve both increased rice yield and remediation, providing a safe, efficient, and environmentally friendly bioremediation method for solving cadmium pollution problems.

[0004] However, the following bottlenecks still exist in the process of transforming this superior strain into large-scale practical applications:

[0005] 1. High production costs: Traditional laboratory cultures often use synthetic media such as NB and LB, which are costly and difficult to meet the economic requirements for large-scale agricultural applications. Finding inexpensive and efficient industrial byproducts to replace traditional carbon and nitrogen sources to achieve high-density fermentation of CML2 is a prerequisite for its industrialization. The use of molasses, an industrial byproduct, as a carbon source for high-density fermentation and its application in the industrial production of *Copper-bacterium* strains is not clearly documented in existing patent literature.

[0006] 2. Insufficient stability and shelf life of microbial agents: Liquid microbial agents often present challenges in storage and transportation, as well as risks of viable cell count decline and contamination. Although general-purpose freeze-drying protectants often contain components such as skim milk powder, trehalose, monosodium glutamate, or glycerol, their protective effects are significantly strain-dependent.

[0007] 3. Lack of systematic application evidence for improving the quality of agricultural products: Although some strains are known to have cadmium-reducing effects, there is still a lack of systematic data support and standardized formulation application guidance to determine whether the strains can successfully colonize rice and synergistically achieve the dual goals of "cadmium reduction" and "yield increase" in the complex environment of actual farmland.

[0008] Therefore, developing a CML2 solid microbial agent that is highly active, highly stable, low-cost, and has a significant effect on reducing cadmium and increasing yield is of great practical significance for solving the "last mile" problem of cadmium pollution remediation in farmland. Summary of the Invention

[0009] The technical problem to be solved by this invention is: how to develop a CML2 solid microbial agent with high activity, high stability, low cost and significant cadmium reduction and yield increase effect.

[0010] The technical solution of this invention is: a method for preparing a solid microbial agent with *Bacillus thuringiensis* CML2 as the active ingredient, comprising the following steps:

[0011] (1) The activated and cultured Bacillus thuringiensis CML2 seed culture was inoculated into the high-density fermentation medium at a volume ratio of 5%, with an initial pH of 7.0 and an initial stirring speed of [missing value]. The initial aeration rate was 1.0 vvm; during fermentation, the stirring speed and / or aeration rate were adjusted according to changes in dissolved oxygen to maintain dissolved oxygen above 30%, and fermentation was carried out until the effective viable bacteria count in the fermentation broth was (1.3±0.2)×10¹² CFU / mL; the preservation number of the *Bacillus thuringiensis* CML2 was CCTCC NO: M 20231365; the composition of the high-density fermentation medium was: 5 g / L sugarcane molasses, 10 g / L peptone and 10 g / L NaCl, prepared with a pH of 7.0–7.2;

[0012] (2) The fermentation broth from step (1) is placed at 4 ℃. Centrifuge for 10 min, discard the supernatant, wash the bacterial cells twice with sterile physiological saline, and collect the concentrated wet bacterial blocks.

[0013] (3) Mix the wet bacterial blocks with the protectant at a ratio of 1 g: 1.5 mL and equilibrate at 37 °C for 30 min; then freeze in liquid nitrogen for 1-2 min and transfer to a vacuum freeze dryer for sublimation drying for 18-24 h until the moisture content is ≤5%; the protectant consists of: 150 g / L skim milk powder, 100 g / L trehalose and 5 g / L sodium glutamate.

[0014] Furthermore, the mass percentage of the total fermentable sugars in the sugarcane molasses in step (1) is ≥45%.

[0015] Furthermore, the seed culture is prepared as follows: a single colony is picked from *Bacillus cereus* CML2 stored at low temperature, inoculated into 100 mL of NB liquid medium, and incubated at 30 °C. Shaking culture until The value was 1.0 ± 0.2, resulting in activated seed solution.

[0016] The solid microbial agent prepared by the above method is used to reduce cadmium accumulation in brown rice and increase the thousand-grain weight of rice.

[0017] Furthermore, the method of application is as follows: reconstitute the solid microbial agent to a viable bacterial concentration. The bacterial suspension was prepared by soaking surface-sterilized rice seeds at 25–30 °C in the dark for 48 h, and replacing the suspension with an equal volume of fresh bacterial suspension after 24 h of soaking.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The solid microbial agent prepared by this invention has an initial effective viable count of (1.2–1.8) × 10¹² CFU / g (based on dry powder mass); after storage at 4℃ in the dark for 180 days, the measured effective viable count is ≥ The number of viable bacteria was measured after storage at 25 ℃ in the dark for 180 days. The CFU / g concentration meets the requirements for effective viable bacteria count in powder form as specified in GB 20287-2006 "Agricultural Microbial Inoculants". This inoculant, applied through seed soaking or soil application, reduces cadmium accumulation in rice grains and increases rice yield, resulting in a 46.2% reduction in cadmium in brown rice and a 10.8% increase in thousand-grain weight in the field. Attached Figure Description

[0020] Figure 1 The effect of 13 culture medium formulations on the viable count of CML2 fermentation.

[0021] Figure 2 The effect of different protective agent formulations on the lyophilization survival rate of CML2, and a comparison of the properties of products containing glycerol (A: viscous lumps; B: loose powder).

[0022] Figure 3 Decline curves of viable bacteria count in solid bacterial powder under storage conditions of 4 ℃ and 25 ℃ (0–180 days, n=3).

[0023] Figure 4 Comparison of genus-level relative abundance of endophytic bacterial communities in rice seeds between CM and CK groups (16S rRNA sequencing, n=3).

[0024] Figure 5Comparison of α-diversity index of endophytic bacterial community in rice seeds between CM and CK groups. Box plots show the inter-group differences (n=3) of the four indices: Chao1, Observed_species, Shannon, and Simpson. * p<0.05; ** p<0.01; *** p<0.001.

[0025] Figure 6 Phenotypic representations of rice seedlings in the CML2 solid microbial agent soaking treatment group and the sterile water control group (left: CK; right: CML2).

[0026] Figure 7 Brown rice Cd content and thousand-grain weight box plots for the solid inoculant group and the sterile water control group in the field trial (A: brown rice Cd content; B: thousand-grain weight). Detailed Implementation

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.

[0028] The *Cupriavidus metallidurans* CML2 described in this invention was deposited at the China Center for Type Culture Collection (CCTCC) on July 24, 2023, with accession number CCTCC NO: M 20231365. It has also been disclosed in CN117229951A.

[0029] The culture medium components involved in the examples are as follows:

[0030] NB medium preparation: 3 g beef extract, 10 g peptone, 10 g NaCl, adjust pH to 7.2, add water to 1L.

[0031] Preparation of NA medium: Add 17g of agar to NB medium to make NA medium.

[0032] MB medium: Weigh 10.0 g of peptone, 3.0 g of maltose, and 10.0 g of sodium chloride (NaCl), dilute with water to 1 L, and adjust the pH to 7.2–7.4.

[0033] DB medium: Weigh 10.0 g of peptone, 3.0 g of soluble starch, and 10.0 g of sodium chloride (NaCl), dilute with water to 1 L, and adjust the pH to 7.2–7.4.

[0034] GB medium: Weigh 10.0 g of peptone, 3.0 g of glucose, and 10.0 g of sodium chloride (NaCl), dilute with water to 1 L, and adjust the pH to 7.2–7.4.

[0035] ND medium: Weigh 10.0 g soybean meal powder, 3.0 g beef extract, and 10.0 g sodium chloride (NaCl), dilute with water to 1 L, and adjust the pH to 7.2–7.4.

[0036] MD medium: Weigh 10.0 g soybean meal powder, 3.0 g maltose, and 10.0 g sodium chloride (NaCl), dilute with water to 1 L, and adjust the pH to 7.2–7.4.

[0037] DD medium: Weigh 10.0 g soybean meal powder, 3.0 g soluble starch, and 10.0 g sodium chloride (NaCl), dilute with water to 1 L, and adjust the pH to 7.2–7.4.

[0038] GD medium: Weigh 10.0 g soybean meal powder, 3.0 g glucose, and 10.0 g sodium chloride (NaCl), dilute with water to 1 L, and adjust the pH to 7.2–7.4.

[0039] NL medium: Weigh 10.0 g of ammonium sulfate, 3.0 g of beef extract, and 10.0 g of sodium chloride (NaCl), dilute with water to 1 L, and adjust the pH to 7.2–7.4.

[0040] ML medium: Weigh 10.0 g ammonium sulfate, 3.0 g maltose, and 10.0 g sodium chloride (NaCl), dilute with water to 1 L, and adjust the pH to 7.2–7.4.

[0041] DL medium: Weigh 10.0 g of ammonium sulfate, 3.0 g of soluble starch, and 10.0 g of sodium chloride (NaCl), dilute with water to 1 L, and adjust the pH to 7.2–7.4.

[0042] GL medium: Weigh 10.0 g of ammonium sulfate, 3.0 g of glucose, and 10.0 g of sodium chloride (NaCl), and dilute with water to 1 L, adjusting the pH to 7.2–7.4.

[0043] All 13 comparative culture media were autoclaved at 121 °C for 20 min and then cooled before use.

[0044] Example 1: Optimization of high-density fermentation medium for Bacillus thuringiensis CML2

[0045] This embodiment aims to screen inexpensive culture media suitable for large-scale production and capable of increasing cell density. The specific operating steps are as follows:

[0046] (1) Seed liquid preparation

[0047] Single colonies were picked from cryopreserved CML2 and inoculated into 100 mL of NB liquid medium at 30 °C. Shaking culture for 20 h to This yields activated seed liquid.

[0048] (2) Culture medium screening experiment (comparison of 13 formulations)

[0049] To compare the effects of different culture media on the viable cell count of CML2 fermentation, this experiment included an NB control group, 11 conventional culture media groups, and one molasses culture media group. The 11 conventional culture media groups were designated MB, DB, GB, ND, MD, DD, GD, NL, ML, DL, and GL, with their specific compositions described in the aforementioned culture medium formulations. The molasses culture media group, designated TM, had a formulation of 5 g / L cane molasses, 10 g / L peptone, and 10 g / L NaCl, with an initial pH of 7.0. All groups were inoculated with 5% (v / v) CML2 seed culture and incubated at 30℃. After shaking culture for 24 h, the final culture solution was serially diluted and plated before the number of viable bacteria was determined.

[0050] Experimental results are as follows Figure 1 As shown, the effective viable count in the TM group was 1.52 × 10¹² CFU / mL, significantly higher than that in the NB group and all conventional culture medium groups. Among the conventional culture medium groups, the DD group had the highest effective viable count. CFU / mL; the effective viable count in the TM group was approximately 27.2 times that of the DD group. The effective viable counts in the NL, ML, DL, and GL groups, using ammonium sulfate as the nitrogen source, were... The CFU / mL concentration was lower than that of other organic nitrogen source culture media.

[0051] (3) Optimization of fermentation parameters

[0052] To verify the applicability of TM medium under fermenter scale-up conditions, TM medium was prepared separately and loaded into a 20L fully automated fermenter. The CML2 seed culture prepared in step (1) was directly inoculated into the TM medium in the fermenter at an inoculation rate of 5% (v / v), with the inoculation time being taken as the fermentation start time.

[0053] The fermentation temperature was 30 ℃, the initial pH of the culture medium was 7.0, and the initial stirring speed was [missing value]. The aeration rate was 1.0 vvm. During fermentation, the stirring speed and / or aeration rate were adjusted according to changes in dissolved oxygen to maintain dissolved oxygen above 30%. After 24 h of culture from inoculation, the effective viable count of CML2 in the final fermentation broth was (1.3±0.2)×10¹² CFU / mL, determined by serial dilution and plate counting.

[0054] Example 2: Preparation of CML2 solid bacterial powder and screening of compound freeze-drying protectant formulation

[0055] (1) Preparation of concentrated wet CML2 cells

[0056] Measure 4 L of the CML2 molasses fermentation broth prepared in Example 1 and incubate it at 4 °C. Centrifuge for 10 min under the specified conditions and discard the supernatant. Resuspend the bacterial cells in sterile physiological saline and wash twice. After centrifugation again, collect approximately 15 g of concentrated wet bacterial blocks, which have a water content of approximately 80%.

[0057] When screening protectant formulations, take concentrated wet bacterial blocks from the same batch or parallel batches prepared under the same fermentation conditions, mix them thoroughly, and then group them into equal amounts to minimize the impact of different batches of bacterial cell states on freeze-drying survival rate.

[0058] (2) Design of compound freeze-drying protectant formulation

[0059] This embodiment is based on a protective system composed of skim milk powder and trehalose. By comparing the reduction of components and the addition of glycerol, the effects of skim milk powder, trehalose, monosodium glutamate, sucrose, ascorbic acid and glycerol on the survival rate, lyophilized powder properties and reconstitution properties of CML2 freeze-dried products are investigated.

[0060] Each protective agent was prepared with distilled water, with a total volume of 15 mL. The specific groupings are as follows.

[0061]

[0062] Experimental groups 1-7 did not contain glycerol. Additionally, 0.75 g of glycerol was added to each of the formulations in experimental groups 1-7 in parallel to create corresponding control groups containing glycerol, which were used to evaluate the effects of glycerol on freeze-drying survival rate, powder properties, and reconstitution performance.

[0063] (3) Freeze-drying and determination of freeze-dried survival rate

[0064] Each group of concentrated wet mycelium blocks was mixed with the corresponding cryoprotectant solution at a ratio of 1 g:1.5 mL (mass of wet mycelium block to volume of cryoprotectant solution), and equilibrated at 37 °C for 30 min. The equilibrated mycelium-crystal protectant mixture was dispensed into sterile freeze-drying trays, flash-frozen in liquid nitrogen for 1–2 min, and then transferred to a vacuum freeze dryer for sublimation drying for 18–24 h. The drying endpoint was defined as a moisture content of no more than 5% in the freeze-dried mycelium powder.

[0065] Samples were taken before and after lyophilization, serially diluted, and spread onto NA solid medium. The samples were incubated at 30 °C for 24 h, and colony counts were performed. Each group had three independent replicates. Results are expressed as mean ± standard deviation.

[0066] Freeze-dried survival rate is calculated using the following formula:

[0067] Freeze-dried survival rate (%) = Total number of viable bacteria in the freeze-dried sample ÷ Total number of viable bacteria in the sample before freeze-drying × 100%.

[0068] Simultaneously observe the powder morphology and agglomeration of the freeze-dried product, and add an equal amount of freeze-dried bacterial powder to a specified volume of sterile water, recording the time required for the bacterial powder to be completely dispersed and form a uniform bacterial suspension, in order to evaluate its reconstitution performance.

[0069] The current results show that the freeze-dried survival rate of CML2 in the NC group without the protective agent was only 8.2%±2.1%; the freeze-dried survival rate of experimental group 1 (with the skim milk powder removed) was 34.5%±4.3%; the freeze-dried survival rate of the Full-CP group was 80.1%±5.2%; and the freeze-dried survival rate of experimental group 7 was 82.4%±4.8%. Experimental group 7, despite reducing the three components of sucrose, ascorbic acid, and glycerol, still maintained a freeze-dried survival rate similar to that of the Full-CP group, while obtaining relatively ideal powder properties and reconstitution performance.

[0070] The bacterial powder obtained in experimental group 7 was loose and porous, and could be completely reconstituted within 30 seconds. The freeze-dried survival rate of the glycerol-containing control group corresponding to experimental group 7 was 68.3% ± 7.4%, a decrease of 14.1 percentage points compared to experimental group 7; its freeze-dried product was viscous and clumped, and could not be properly reconstituted. The product characteristics of experimental group 7 and its glycerol-containing control group are shown below. Figure 2 .

[0071] The above results indicate that, under the ternary protective agent system and freeze-drying conditions used in this embodiment, the addition of glycerol failed to further improve the freeze-drying survival rate of CML2 and was not conducive to the formation of loose, easily resoluble solid bacterial powder. This phenomenon may be related to the hygroscopicity of glycerol and its influence on the formation of porous solid structures during the freeze-drying process.

[0072] (5) Optimized formula and preparation of solid bacterial powder

[0073] Taking into account freeze-drying survival rate, protectant composition, powder properties, and reconstitution performance, experimental group 7 was selected as the preferred protectant formulation for subsequent experiments. This protectant consists of 150 g / L skim milk powder, 100 g / L trehalose, and 5 g / L monosodium glutamate, and does not contain sucrose, ascorbic acid, or glycerol.

[0074] According to the ratio of 1 g to 1.5 mL of wet mycelium block mass to protective agent solution volume, CML2 concentrated wet mycelium block was mixed with the above-mentioned ternary composite protective agent, equilibrated at 37 ℃ for 30 min, and then freeze-dried in liquid nitrogen for 1-2 min under vacuum for 18-24 h to obtain CML2 solid mycelium powder with a moisture content of no more than 5%.

[0075] After serial dilution and plate counting, the initial viable count of the obtained bacterial powder was (1.5±0.2)×10¹²CFU / g (n=3), and the freeze-dried survival rate was 82.4%±4.8%. The obtained bacterial powder was loose and porous, without obvious viscous clumps, and could be completely reconstituted within 30 s.

[0076] Example 3: Evaluation of shelf-life stability of solid microbial agents

[0077] Solid bacterial powder prepared using the ternary composite protective agent formulation described in Experimental Group 7 of Example 2 was weighed and dispensed into 10.0 g tubes, sealed with sealing film, for a total of 24 tubes: 12 tubes each for the 4 ℃ refrigerated group (60% ± 10% relative humidity, protected from light) and the 25 ℃ room temperature group (60% ± 10% relative humidity, protected from light). Three tubes were taken at days 0, 30, 90, and 180 to test the effective viable bacteria count, moisture content, and pH of the compound. Results are as follows: Figure 3 As shown:

[0078]

[0079] GB 20287-2006 "Agricultural Microbial Inoculants" requires a minimum number of viable bacteria in powder formulations. CFU / g. The viable bacterial counts in both the 4℃ and 25℃ groups met the requirement on day 180; the actual measured count in the 4℃ group was [missing value]. CFU / g, measured at 25 ℃: CFU / g. Moisture content change ≤0.5 percentage points during storage, pH change of reconstituted solution ≤0.2, and no powder agglomeration. Based on existing measured data, this application sets the directly verifiable shelf life at 180 days; longer periods should be claimed only after completing measurements at the corresponding time points.

[0080] Example 4: Verification of endophytic colonization of CML2 in rice seeds

[0081] This embodiment aims to verify the endophytic colonization ability of the solid bacterial agent prepared by this invention, after being compounded into a bacterial suspension, within rice seeds and its impact on the internal microbial community structure of the rice seeds. Specific experimental steps and results are as follows:

[0082] (1) Preparation of bacterial suspension and experimental design

[0083] The solid bacterial powder obtained in Example 2 was dissolved in sterile pure water and adjusted... The viable bacterial concentration was confirmed to be approximately 0.50 by plate counting. CFU / mL, store at 4 ℃ for later use, and complete the soaking process within 2 hours.

[0084] This experiment consisted of two treatment groups, each with three independent biological replicates. Approximately 500 healthy rice seeds (Jinxiangyu No. 1 variety) of uniform size, plump grains, without damage or mold were precisely selected for each group. The control group (CK group) used sterile pure water as the soaking medium, ensuring complete seed immersion. The experimental group (CM group) used the aforementioned CML2 sterile pure water bacterial suspension as the soaking medium, with soaking conditions (volume, temperature, and time) identical to the control group.

[0085] (2) Microbial community analysis

[0086] After soaking, rice seeds from each group were collected, and total genomic DNA of prokaryotes was extracted. Phylogenetic and taxonomic analyses of the internal microbial communities of rice seeds before and after soaking were performed at the genus level using 16S rRNA V3-V4 region high-throughput sequencing technology. Results are as follows: Figure 4 As shown, there were significant differences in the endophytic bacterial community structure between the CK and CM groups of rice. *Cupriavidus* was almost undetectable in the CK group (relative abundance < 0.01%), while it significantly increased to 0.55% ± 0.04% in the CM group (p < 0.001), ranking 14th, confirming that CML2 successfully colonized the rice seed. Simultaneously, the relative abundance of groups such as *Bellaquia* and *Pantoea* in the CM group also showed a corresponding increase, rising from approximately 0.02% and 0.17% in the CK group to 0.45% and 0.66%, respectively, suggesting that CML colonization may indirectly promote the proliferation of specific indigenous groups through metabolite secretion or microenvironmental alterations. In both groups of samples, the proportion of unclassified microbiota (Others) remained at approximately 74%, indicating that the introduction of exogenous strains did not cause drastic disturbance to the overall community. Instead, it confirmed that CML2 could successfully enter the rice seed and achieve endophytic colonization within the existing microbiome framework. These results provide microbiome evidence for the systematic migration and subsequent functional role of CML2 in rice plants.

[0087] (3) Analysis of changes in α diversity

[0088] The results are as follows Figure 5As shown, the consistency among replicates in the CK group was good. After soaking in CML2, the Chao1, Observed_species, Shannon, and Simpson indices in the CM group all decreased, indicating that the soaking treatment had a stable and repeatable remodeling effect on the rice seed endophytic microbiota. Given that the relative abundance of the genus Cupriavidus is approximately 0.55%, the above changes may be related to the alteration of the physicochemical microenvironment and indirect interactions among the microbiota during the soaking process. A decrease in diversity itself cannot be directly equated with a beneficial ecological effect; however, considering the consistency among replicates, the lack of decrease in the final seed germination rate, the lack of inhibition of early seedling growth, and subsequent field results, it can be confirmed that no significant growth safety risk was observed under the conditions of this embodiment.

[0089] Example 5: Effects of CML2 solid inoculant seed soaking on chlorophyll content and root biomass of rice seedlings

[0090] This embodiment uses rice seedlings obtained by reconstitution and soaking in CML2 solid bacterial agent as described in Example 4, and sterile water control seedlings at the same time, to further evaluate its effect on photosynthetic pigments in seedling leaves and root biomass.

[0091] (1) Chlorophyll determination

[0092] Seedlings from the control group (CK) and the CML2 inoculant treatment group were randomly selected at the same time. Leaves were cut into 2–3 mm wide strips and immersed in 80% acetone or 95% ethanol, respectively, and extracted at room temperature in the dark for 48 h until the leaves were completely decolorized. The absorbance of the acetone extract was measured at 645 nm and 663 nm, and the absorbance of the ethanol extract was measured at 649 nm and 665 nm. The absorbance at 470 nm was also measured to calculate the carotenoid content.

[0093]

[0094] The results from the two extraction methods were consistent: the total chlorophyll in the CML2 treatment group increased from 0.46 mg / g to 0.88 mg / g and from 0.89 mg / g to 1.10 mg / g, respectively, with increases of 0.42 mg / g and 0.21 mg / g; the carotenoids measured by the acetone method increased from 0.02 mg / g to 0.04 mg / g, while the carotenoids measured by the ethanol method remained unchanged.

[0095] (2) Root dry weight determination

[0096] Thirty rice seedlings of the same age were randomly selected from both the control group and the CML2 treatment group. Root systems were isolated, dried at 60 ℃ for 12 hours, and then weighed. The root dry weight of the CML2 treatment group was approximately 1.8 times that of the control group; representative plant phenotypes are shown below. Figure 6 .

[0097] The above results indicate that reconstituted CML2 solid microbial agent can promote root biomass accumulation and increase total chlorophyll content in leaves during the seedling stage, thereby providing early physiological support for subsequent nutrient absorption and grain filling.

[0098] Example 6: Field application verification of CML2 microbial agent in cadmium-contaminated paddy fields

[0099] This embodiment aims to verify the effectiveness of the *Cupriavidus metallidurans* CML2 inoculant described in this invention in actual cadmium-contaminated paddy fields through a field plot trial, and to systematically evaluate its comprehensive impact on rice seed germination, early growth, cadmium accumulation in brown rice at maturity, and rice yield. Specific experimental methods and results are as follows:

[0100] (1) Experimental site and field trial design

[0101] Honda selected a cadmium-contaminated farmland in central China (specifically, Lingmi Township, Jiangxia District, Wuhan City) as the test site. The soil type in the test area was conventional paddy soil, and the basic physicochemical properties were measured as follows: total cadmium (Cd) content in the soil was 0.578 mg / kg, pH value was 7.0–7.3, and overall fertility was at the local conventional level.

[0102] The total area of ​​the field trial was 50 mu (approximately 3.3 hectares). The experimental field was divided into a treatment group (treated with CML2 inoculant) and a control group (CK group, no inoculant applied), with three replicates per group, each group covering approximately 25 mu (approximately 1.6 hectares). To strictly ensure the accuracy of the experimental data, isolation rows approximately 0.3 m wide were set between each replicate plot, and the experimental group and the control group were physically separated by 0.7 m wide ridges to completely avoid inoculant diffusion and water cross-contamination between plots.

[0103] (2) Preparation, quality control and field application of single-strain agents

[0104] Solid bacterial powder was produced on a large scale using the molasses fermentation-freeze-drying process described in Examples 1-2 (20 L fermenter, 4 batches, totaling approximately 60 L of fermentation broth), and dissolved in sterile water to prepare... CFU / mL bacterial suspension. Purity verification: The bacterial suspension was serially diluted and spread onto LB agar plates. After 24 hours of incubation, no contaminating bacteria were observed, indicating that the bacterial agent was of acceptable purity. The bacterial suspension should be prepared fresh and used immediately, and applied within 4 hours.

[0105] (3) Effects of soaking treatment on rice seed germination

[0106] Seed soaking treatment: Rice seeds were surface disinfected using conventional methods and rinsed with sterile water. The experimental group was soaked in a CML2 bacterial suspension, while the control group was soaked in an equal volume of sterile water. Both groups were kept at 28 ℃ in the dark for 48 h, with the solution changed every 24 h. At 24 h of soaking, the white sprouting rate in both groups was 45%–55%, with no significant difference (p>0.05). At 48 h of soaking, the white sprouting rate in the treatment group was 81.2%, and in the control group it was 82.3%, with no significant difference (p>0.05). After 48 h of germination, the final germination rate in both groups was 100%. On the 7th day of hydroponics, no significant differences were observed in early phenotypes such as plant height, root length, and leaf unfolding between the two groups (p>0.05), indicating that this soaking condition did not have any adverse effects on the early development of rice.

[0107] (4) Effects of microbial agent application on cadmium content and thousand-grain weight (yield) of brown rice

[0108] Brown rice was harvested using a five-point sampling method at maturity. After drying at 65 ℃ to constant weight, passing through a 100-mesh sieve, and microwave digestion, the cadmium content in the brown rice was determined by ICP-MS (limit of detection 0.001 mg / kg, recovery rate 95%–105%). The results are as follows: Figure 7 As shown in Figure A.

[0109] The cadmium content in brown rice of the control group (CK) was 0.026 ± 0.004 mg / kg, while that of the CML2 microbial agent treatment group was 0.014 ± 0.003 mg / kg. Both groups were lower than the cadmium content limit for rice specified in GB 2762-2022 "National Food Safety Standard - Limits of Contaminants in Food" (≤0.2 mg / kg). Compared with the control group, the cadmium content in brown rice of the microbial agent treatment group decreased by approximately 46.15%, and the difference was statistically significant (p<0.05). The results indicate that even in an environment with relatively low soil cadmium background values ​​(0.578 mg / kg total Cd in the soil of this experimental site), CML2 microbial agent treatment can still significantly reduce cadmium accumulation in rice grains, further improving the safety and quality assurance level of rice within the national standard limits.

[0110] The results of the thousand-grain weight test of brown rice are as follows: Figure 7 As shown in Figure B. The control group had a dry weight of 19.5 ± 1.2 g, while the CML2 inoculant treatment group had a dry weight of 21.6 ± 0.8 g, an increase of approximately 10.77% compared to the control group (p < 0.05). Combined with the root dry weight and chlorophyll response observed in Example 6, these results support the possibility that CML2 treatment may promote nutrient absorption and grain filling by improving root growth and photosynthetic pigment accumulation; however, the specific mechanism of action still requires further experimental verification.

[0111] Based on the above results, under the soil background and application conditions of this embodiment, soaking seeds after reconstitution of the specific CML2 solid microbial agent did not inhibit rice germination and early growth, and reduced cadmium accumulation in brown rice by about 46.15% and increased thousand-grain weight by about 10.77%, demonstrating a synergistic effect of cadmium reduction and weight gain.

Claims

1. A method for preparing a solid microbial inoculant using *Bacillus thuringiensis* CML2 as the active ingredient, characterized in that, Includes the following steps: (1) The activated and cultured Bacillus thuringiensis CML2 seed culture was inoculated into the high-density fermentation medium at a volume ratio of 5%, with an initial pH of 7.0 and an initial stirring speed of 300 r•min. -1 The initial aeration rate was 1.0 vvm. During fermentation, the stirring speed and / or aeration rate were adjusted according to changes in dissolved oxygen to maintain dissolved oxygen above 30%. Fermentation continued until the effective viable cell count in the fermentation broth reached (1.3 ± 0.2) × 10⁻⁶. 12 CFU / mL; the preservation number of the copper-loving bacillus CML2 is CCTCC NO: M 20231365; the composition of the high-density fermentation medium is: 5 g / L sugarcane molasses, 10 g / L peptone and 10 g / L NaCl, prepared with a pH of 7.0–7.2; (2) The fermentation broth from step (1) was heated at 4 °C and 4000 r·min. -1 Centrifuge for 10 min, discard the supernatant, wash the bacterial cells twice with sterile physiological saline, and collect the concentrated wet bacterial blocks. (3) Mix the wet bacterial blocks with the protectant at a ratio of 1 g: 1.5 mL and equilibrate at 37 °C for 30 min; then freeze in liquid nitrogen for 1-2 min and transfer to a vacuum freeze dryer for sublimation drying for 18-24 h until the moisture content is ≤5%; the protectant consists of: 150 g / L skim milk powder, 100 g / L trehalose and 5 g / L sodium glutamate.

2. The preparation method according to claim 1, characterized in that, In step (1), the total mass percentage of fermentable sugars in the sugarcane molasses is ≥45%.

3. The preparation method according to claim 1, characterized in that, The seed culture was prepared as follows: a single colony was picked from *Bacillus cereus* CML2 stored at low temperature and inoculated into 100 mL of NB liquid medium, incubated at 30 ℃ and 200 r•min. -1 Shaking culture to OD 600 The value was 1.0 ± 0.2, resulting in activated seed solution.

4. The application of the solid microbial inoculant obtained by the preparation method according to any one of claims 1-3 in reducing cadmium accumulation in brown rice and increasing the thousand-grain weight of rice.

5. The application according to claim 4, characterized in that, The method of application is as follows: the solid microbial agent is reconstituted to a viable bacterial concentration of (1.0–3.0) × 10⁻⁶. 9 CFU / mL bacterial suspension; surface-sterilized rice seeds were soaked in the dark at 25–30℃ for 48 h, and the same volume of fresh bacterial suspension was replaced after 24 h of soaking.

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