Application of Spirodela and microorganism in repairing cadmium contaminated water and reducing cadmium content in rice grain in cadmium contaminated paddy field

Abstract

The application is the application of Spirodela and microorganisms in repairing cadmium-polluted water bodies and reducing cadmium content in cadmium-polluted paddy field rice grains. The specific steps are as follows: first, prepare Oscillospiraceae bacterial suspension, Enterobacter bacterial suspension and Pantoea bacterial suspension respectively; then mix the three kinds of bacterial suspensions in proportion to obtain a mixed bacterial suspension; then add the mixed bacterial suspension and Spirodela prepared in proportion to the cadmium-polluted water bodies to be repaired or the cadmium-polluted paddy fields to be repaired, and the Spirodela and the microorganisms form a "Spirodela-functional bacterial community" symbiotic system; the microorganisms further transport cadmium to the leaves by adsorption, transformation, leaf surface sedimentation or transpiration; finally, the Spirodela is concentrated for treatment, thereby repairing the cadmium-polluted water bodies or reducing the cadmium content in the cadmium-polluted paddy field rice grains. Through the application, the cadmium-polluted water bodies can be better repaired, and the cadmium content in the cadmium-polluted paddy field rice grains can be effectively reduced.

Description

Application of the combination of duckweed and microorganisms in the remediation of cadmium-contaminated water bodies and the reduction of cadmium content in rice grains from cadmium-contaminated paddy fields Technical Field

[0001] This invention belongs to the field of phytoremediation technology, specifically the application of duckweed and microorganisms in the remediation of cadmium-polluted water bodies and the reduction of cadmium content in rice grains from cadmium-polluted paddy fields. Background Technology

[0002] In recent years, with the continuous acceleration of industrialization, pollution problems caused by heavy metals such as cadmium (Cd), mercury (Hg), and lead (Pb) have become increasingly serious. Among them, cadmium pollution is particularly severe, widely distributed in water bodies and soil, and has become one of the most serious ecological problems globally. Due to its high solubility in water, strong chemical stability, and extremely long half-life (30-70 years), cadmium has high mobility and bioavailability in water bodies, and can migrate and accumulate widely between water bodies, soil, and organisms, undergoing biomagnification through the food chain. This leads to stunted growth and even death of plants and animals in the environment. For plants, cadmium can inhibit photosynthesis, reduce the plant's ability to absorb nutrients, inhibit the activity of antioxidant enzymes, and interfere with its normal metabolic processes, resulting in slow plant growth, and in severe cases, wilting or even death. For example, cadmium pollution can exacerbate leaf cell damage and weaken rehydration capacity by altering the water relationship of tomato tissues (reducing cell wall stiffness and affecting the turgor pressure loss point) and mineral distribution, thereby increasing the risk of leaf hydraulic system collapse. Cadmium can also enter the human body through the food chain, primarily accumulating in the kidneys and liver. Chronic cadmium exposure is associated with a variety of serious health problems, such as kidney damage and bone demineralization, including Itai-itai disease. Studies have found that cadmium can cause testicular interstitial cell damage by activating the TNF-α / TNFR1 signaling pathway and reactive oxygen species (ROS)-mediated necrotizing apoptosis. Growing rice and vegetables in cadmium-contaminated water poses a serious threat to human health. Therefore, developing economical and safe technologies to remove Cd contaminants is crucial for the long-term health of humans and ecosystems.

[0003] Currently, many physical, chemical, and biological methods are used to remove Cd from water. Physical and chemical methods mainly involve adding adsorbents and neutralizing agents. However, these methods are costly and may cause secondary pollution. Biological methods utilize plants or microorganisms for absorption. Phytoremediation technology is cost-effective and environmentally friendly, and is widely used to remove heavy metals from water bodies, sediments, and soil. However, current biological methods are slow and their mechanisms of action are unclear.

[0004] Therefore, we still need to further develop and utilize this technology. Summary of the Invention

[0005] The purpose of this invention is to address the problems existing in the prior art by providing an application of *Lemna minor* combined with microorganisms in the remediation of cadmium-contaminated water bodies and in reducing the cadmium content of rice grains in cadmium-contaminated paddy fields. The interaction between plants and microorganisms, through secreting organic acids to reduce the bioavailability of heavy metals, inducing plant antioxidant systems, regulating plant hormones, or directly participating in metal ion transport, synergistically responds to and controls Cd uptake in multiple ways, enhancing the host plant's tolerance to adversity and remediation efficiency. Through this application, cadmium-contaminated water bodies can be effectively remediated, and the cadmium content of rice grains in cadmium-contaminated paddy fields can be effectively reduced.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] One of the objectives of this invention is to protect the application of duckweed combined with microorganisms in the remediation of cadmium-contaminated water bodies.

[0008] The second objective of this invention is to protect the application of the combination of duckweed and microorganisms in reducing the cadmium content of rice grains in cadmium-polluted paddy fields.

[0009] Furthermore, in the application of duckweed combined with microorganisms in the remediation of cadmium-contaminated water bodies or in the application of duckweed combined with microorganisms in reducing the cadmium content of rice grains in cadmium-contaminated paddy fields, the mass ratio of duckweed to microorganisms is 1:0.01-0.1. More preferably, the mass ratio of duckweed to microorganisms is 1:0.05.

[0010] Furthermore, in the application of duckweed combined with microorganisms in the remediation of cadmium-polluted water bodies or in the application of duckweed combined with microorganisms in reducing the cadmium content of rice grains in cadmium-polluted paddy fields, the microorganisms include *Streptococcus*, *Enterobacter*, and *Pantotheca*.

[0011] Furthermore, Herbaspirillum, Enterobacter, and Pantoea can be screened and isolated from paddy fields, or they can be purchased. For example, Herbaspirillum can be purchased from the China Microbial Culture Collection Platform (Platform No.: Bio-59026; Preservation No.: CCTCC AB 2018017), Enterobacter can be purchased from the China Microbial Culture Collection Platform (Platform No.: Bio-62157; Preservation No.: CCTCC AB 2013293), and Pantoea can be purchased from the China Microbial Culture Collection Platform (Platform No.: Bio-60429; Preservation No.: CCTCC AB 206575).

[0012] Furthermore, in the application of *Lemna minor* combined with microorganisms in the remediation of cadmium-polluted water bodies or in the application of *Lemna minor* combined with microorganisms in reducing the cadmium content of rice grains in cadmium-polluted paddy fields, the mass ratio of *Lemna minor*, *Enterobacter*, and *Pantotheca* is 2.2-2.8:2.2-2.8:1; more preferably, the mass ratio of *Lemna minor*, *Enterobacter*, and *Pantotheca* is 2.5:2.5:1.

[0013] Furthermore, in the application of *Lemna minor* combined with microorganisms in the remediation of cadmium-contaminated water bodies or in the application of *Lemna minor* combined with microorganisms in reducing the cadmium content of rice grains in cadmium-contaminated paddy fields, the microorganisms exist in the form of a mixed bacterial suspension. The mixed bacterial suspension refers to a mixed bacterial suspension containing *Streptococcus*, *Enterobacter*, and *Pantotheca* strains, with each bacterial suspension containing 10 effective strains. 6 CFU / mL or higher.

[0014] As a preferred embodiment of this application, the specific application method of combining duckweed and microorganisms in the remediation of cadmium-contaminated water includes the following steps:

[0015] First, suspensions of *Streptococcus*, *Enterobacter*, and *Pantotheca* were prepared separately. Then, the three suspensions were mixed in proportion to obtain a mixed bacterial suspension. Next, the mixed bacterial suspension was prepared with *Lemna minor* in proportion (mixed or soaked) and added to the cadmium-polluted water body to be remediated. *Lemna minor* and microorganisms formed a symbiotic system of "Lemna minor-functional microbial community". The microorganisms then transported cadmium to the leaves through adsorption, transformation, leaf surface sedimentation, or transpiration. Finally, the *Lemna minor* was collected and treated centrally (or released with the water for filtration), thereby achieving the remediation of the cadmium-polluted water body.

[0016] Furthermore, in the aforementioned application, the cadmium concentration in cadmium-contaminated water bodies and cadmium-contaminated paddy fields is no higher than 2 μM (10 μM treatment was acceptable in experiments, but it caused significant damage to duckweed). Since existing severe pollution cases have not exceeded this concentration, this method is applicable to cadmium pollution problems encountered in modern agricultural production.

[0017] Furthermore, in the aforementioned application, the cadmium concentration in the remediated water body is ≤0.2276 mg / kg, and the cadmium content in rice grains is 0.01-0.078 mg / kg, which is significantly lower than the standard of 0.20 mg / kg.

[0018] As a preferred embodiment of this application, the application of duckweed and microorganisms in reducing the cadmium content of rice grains in cadmium-polluted paddy fields includes the following steps: First, prepare suspensions of *Streptococcus*, *Enterobacter*, and *Pantotheca* respectively; then mix the three suspensions in a certain proportion to obtain a mixed suspension; next, add the mixed suspension and duckweed in a certain proportion to the cadmium-polluted paddy field to be remediated, where duckweed and microorganisms form a symbiotic system of "duckweed-functional microbial community"; the microorganisms then transport cadmium to the leaves through adsorption, transformation, leaf surface sedimentation, or transpiration; finally, the duckweed is centrally treated (e.g., centralized collection and fermentation, centralized incineration of cadmium with solid sedimentation, and continued return of liquid to the field), thereby reducing the cadmium content of rice grains in cadmium-polluted paddy fields.

[0019] Furthermore, this application can reduce the cadmium content in rice grains from 0.40 mg / kg to <0.20 mg / kg.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1) Under Cd stress, specific functional characteristics such as the nitrate / nitrite transporter NarK, signal transduction mechanisms, and ion channel proteins are enriched. This invention reveals the roles of *Lemna minor* and microorganisms in the response to cadmium stress, providing a theoretical basis for the joint remediation of cadmium-polluted water bodies by plants and microorganisms.

[0022] 2) The method in this invention has good environmental compatibility, low cost, and is easy to promote;

[0023] 3) This invention can effectively remediate cadmium-polluted water bodies.

[0024] 4) The present invention can reduce the cadmium content in rice. Attached Figure Description

[0025] Figure 1 shows the growth status of Spirodela polyrhiza under different cadmium (Cd) concentration stresses;

[0026] Among them, (a, e, i) are the growth phenotypes after 7 days of treatment with 0, 1 and 10 μM Cd, respectively;

[0027] (b–d) are scanning electron microscope images of leaves in the control group (0 μM Cd); (f–h) are scanning electron microscope images of leaves in the 1 μM Cd treatment group; (jl) are scanning electron microscope images of leaves in the 10 μM Cd treatment group; red arrows indicate microorganisms attached to the leaf surface;

[0028] Figure 2 shows the diversity curve of bacterial communities associated with duckweed leaves;

[0029] Among them, (a) species abundance-diversity curve; (b) species dilution curve, where CK, 0 μM Cd; Cd1, 1 μM Cd; Cd10, 10 μM Cd.

[0030] Figure 3 shows the Alpha diversity index of bacterial communities in duckweed leaves under different Cd treatments.

[0031] Wherein, CK, 0 μM Cd; Cd1, 1 μM Cd; Cd10, 10 μM Cd;

[0032] Figure 4 shows the principal coordinate analysis (PCoA) of the bacterial community on the leaves of *Lemna minor* under different Cd concentrations;

[0033] Wherein, CK, 0 μM Cd; Cd1, 1 μM Cd; Cd10, 10 μM Cd;

[0034] Figure 5 shows the taxonomic composition of the microbial community on the leaves of *Lemna minor* under Cd stress.

[0035] The Venn plot shows unique and shared ASVs across treatments. (b-e) Relative abundance of the top 10 taxa at the phylum (b), class (c), order (d), and family (e) levels. (f) Heatmap of the top 30 genera; color bars indicate relative abundance from low (dark red) to high (dark blue). Cd10, 10 μM Cd; Cd1, 1 μM Cd; CK, 0 μM Cd;

[0036] Figure 6 shows the LEfSe analysis of the differential groups between the Cd10 group and the CK group;

[0037] Among them, the linear discriminant analysis (LDA) of LEfSe was used to distinguish from class to species (LDA score>2, P<0.05). (b) The circles radiating outward from the inside of the clade diagram represent the classification level from class to genus. Cd10, 10 μM Cd; CK, 0 μM Cd.

[0038] Figure 7 shows the predicted functional spectrum of bacterial communities in the leaves of *Lemna minor* under Cd stress;

[0039] Among them, (a) enrichment of COG function among different samples. (b) enrichment of KEGG pathway among different samples. Cd10, 10 μM Cd; Cd1, 1 μM Cd; CK, 0 μM Cd. Detailed Implementation

[0040] The specific embodiments of the present invention will be further described in detail below with reference to the examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. The present invention covers all possible alternatives, improvements, and equivalents within the scope of the claims. Specific techniques or conditions not specified in the following examples are conventional techniques or conditions, or techniques or conditions described in the literature in the art, or according to the product manual.

[0041] In this application, all percentages not explicitly stated are mass percentages, i.e., wt%.

[0042] Example 1

[0043] Experiment 1: Study on the growth status of *Lemna minor* leaves under different cadmium concentrations:

[0044] Spirodela polyrhiza leaves were exposed to different cadmium concentrations of 0, 1, and 10 μM to assess their microbial and structural responses.

[0045] *Leptochloa crus-galli* was collected from a paddy field in Shuangliu District, Chengdu, Sichuan Province, and preserved in the laboratory of Chengdu University (No. 001). It was cultured according to existing methods: the leaves were transferred to a 1000 mL glass beaker containing 800 mL of field water and incubated at 25±1℃, with a 16 h light / 8 h dark cycle and a light intensity of 40 μmol·m⁻¹. -2 ·s -1 Under these conditions, the cells were pre-cultured for 3 days. Healthy, uniformly grown leaves were selected for stress experiments. Stress levels of 0, 1.0, and 10.0 µM CdCl2 were set by adding CdCl2 to the culture medium. 2+ Treatment groups. Each treatment was maintained under identical growth conditions for 7 days, with 3 biological replicates for each concentration. Exposure to 0, 1.0, and 10.0 μM Cd was performed. 2+Seven-day-old *Lemna minor* leaves (denoted as CK, Cd1, and Cd10, respectively) were centrifuged at 6000 rpm for 10 minutes to separate each precipitate. The samples were fixed with 2.5% (v / v) glutaraldehyde prepared in 0.1% potassium phosphate buffer, washed with distilled water, dehydrated with a gradient of ethanol, and dried using a freeze dryer. The adaxial surface of the leaves was then placed on a spherical metal column, fixed with tape, and gold was deposited on the surface using a sputtering coating instrument. The surface features and stomata were then observed using a scanning electron microscope at 5 kV. The specific results are shown in Figure 1 and Table 1.

[0046] Table 1. Characteristic parameters of stomata in duckweed leaves under different cadmium concentrations

[0047]

[0048] CK, 0 μM Cd; Cd1, 1 μM Cd; Cd10, 10 μM Cd. Data in the table are expressed as mean ± standard deviation. Different lowercase letters in the same column indicate significant differences between samples (P < 0.05).

[0049] As shown in Figure 1 and Table 1, in the control group (CK), the leaves of *Lemna minor* remained plump and uniformly green (Figure 1a). When exposed to 1 μM Cd (Cd1), some leaves showed slight yellowing (Figure 1e); while when exposed to 10 μM Cd (Cd10), the leaves showed large-area discoloration and obvious wilting (Figure 1i). Scanning electron microscopy (SEM) analysis showed that the stomatal structure of *Lemna minor* leaves changed significantly in a concentration-dependent manner with increasing cadmium concentration. Seven days after cadmium exposure, there was no significant difference in stomatal length among the treatment groups (P>0.05); in contrast, the stomatal width and pore area of ​​the Cd1 and Cd10 groups increased significantly, by 2.02-2.70 times and 2.04-2.46 times, respectively, compared with the CK group (P<0.05). Among them, the average stomatal width (3.38 μm) and pore area (29.81 μm) of the Cd1 group were significantly larger. 2 The largest pore density was observed in the Cd10 group (189.11 pores / mm²), while the highest pore density was observed in the Cd10 group (189.11 pores / mm²). 2 It is worth noting that the pore densities of the Cd1 and Cd10 groups were 3.30 times and 4.21 times that of the CK group, respectively (P<0.05).

[0050] Simultaneously with morphological changes, the colonization of epiphytic microorganisms increased with increasing cadmium concentration. In the CK group, the leaf surface was intact with slight wrinkles, and the stomata were small in size with few microorganisms (bd in Figure 1). Under 1 μM Cd treatment, shallow grooves appeared on the leaf edges, the stomata enlarged, and the amount of attached microorganisms slightly increased (fh in Figure 1). Under 10 μM Cd treatment, the grooves on the leaf surface deepened, local structures were damaged, and the edges were slightly wrinkled. While the number of stomata remained relatively high, adhesive blockages frequently appeared within the pores, and the leaf surface was covered with a dense bacterial layer (jl in Figure 1). These results indicate that cadmium stress can simultaneously reshape the stomatal structure of *Lemna minor* leaves and promote the enrichment of epiphytic microorganisms.

[0051] Experiment 2: Analysis of Microbial Composition of Duckweed Leaves under Cadmium Stress

[0052] To elucidate the effects of cadmium stress on microbial colonization of *Lemna minor* leaves, high-throughput 16S rRNA gene sequencing was performed on bacteria from leaf surfaces exposed to 0, 1, and 10 μM Cd. A total of 514 species belonging to 26 phyla, 64 classes, 125 orders, 206 families, and 406 genera were detected. Specific results are shown in Figure 2. The species abundance curve (Figure 2a) reflects species richness and evenness: a larger horizontal span indicates higher species richness; a smoother curve indicates higher community evenness. In this study, the horizontal zigzag lines of each group of samples were relatively wide and eventually flattened, indicating a homogeneous and rich sample composition. The dilution curve (Figure 2b) further shows that the number of observed species gradually increased with increasing sequencing depth, and the sparse curve tended to stabilize, indicating a reasonable amount of data.

[0053] Experiment 3: Alpha diversity analysis of bacterial communities on duckweed leaves

[0054] The richness and evenness of leaf-related bacterial communities were assessed using the α-diversity index (see Figure 3 for details). Observed species, Chao1 index, and ACE index reflected community richness; the results showed that the CK group had the highest richness, followed by the Cd1 group, while the Cd10 group had the lowest. Compared to the CK, both the Cd1 and Cd10 groups showed a decrease in observed species and Chao1 index. The Shannon and Simpson indices reflected inter-community species diversity; the results showed that the Cd1 treatment had the highest microbial community diversity, even higher than the CK treatment, indicating that moderate cadmium stress can promote a more even community structure. In summary, cadmium exposure reduced the overall richness of the bacterial community in Drosophila leaves but increased community evenness, indicating a shift towards a more balanced microbiome structure.

[0055] Experiment 4: Beta diversity analysis of bacterial communities on duckweed leaves

[0056] Figure 4 shows the differences in microbial community species diversity among different samples. Principal coordinate analysis (PCoA) was used to measure the differences in microbial community structure similarity among sample groups. The figure shows that the PCoA1 and PCoA2 axes explained 31.07% and 23.58% of the changes in bacterial community composition, respectively. The Cd10 treatment group showed good separation from the control group (Figure 4), indicating a difference between the bacterial community on untreated *Lemna minor* leaves and that under 10 μM cadmium treatment. 10 μM Cd stress altered the structure of the *Lemna minor* bacterial community, leading to differentiation compared to the control group.

[0057] Experiment 5: Microbial community structure under Cd treatment

[0058] To investigate the effects of different concentrations of Cd treatment on the leaf microbial community of *Symplocos rubra*, we used 16S rRNA sequences to study the leaf microbial community of *Symplocos rubra* samples. 1473 ASVs obtained through DADA2 noise reduction were used for species classification. Venn analysis (Figure 5a) showed that the CK, Cd1, and Cd10 groups had 400, 246, and 212 unique ASVs, respectively, and 324 shared ASVs across the three groups, indicating that the *Symplocos rubra* leaf microbial community contains both core taxa and treatment-specific taxa.

[0059] At the phylum level (Figure 5b), Cyanobacteria were dominant in the CK group, but decreased by 23% (to 46.51%) in the Cd1 group and remained at a low level in the Cd10 group. Proteobacteria increased significantly under cadmium stress: from 32.6% in the CK group to 46.26% (an increase of 42%) in the Cd1 group and 42.68% (an increase of 31%) in the Cd10 group. The fourth most dominant phylum, Bacteroidota, had a significantly lower relative abundance in the Cd10 group than in the CK group (P<0.05).

[0060] At the class level (Figure 5c), significant variations were observed within the Proteobacteria phylum: Cyanobacteria accounted for an average of 53.4% ​​of all samples, followed by Gammaproteobacteria (25.2%), Alphaproteobacteria (8.8%), and Betaproteobacteria (6.5%). Notably, the relative abundance of Gammaproteobacteria in the Cd10 group increased to 36.2%, which was 1.3 times that of the CK group (15.96%) (P<0.05).

[0061] At the order level (Figure 5d), Chloroplasts and Burkholderiales remained highly abundant in all treatment groups. The Cd10 group specifically enriched Enterobacterales (P<0.05) while significantly reducing the abundance of Sphingomonadales (P<0.05). At the family level (Figure 5e), Methylophilaceae was the most dominant family overall, showing a trend of "Cd10>Cd1>CK"; Comamonadaceae and Sphingomonadaceae ranked second and third, respectively.

[0062] At the genus level, the heatmap of the top 30 dominant genera (Figure 5f) shows significant changes in community composition with increasing cadmium concentration. In the Cd10 group, the relative abundance of *Methylophilus* reached 20.03% (compared to 8.44% in the CK group) and 13.75% in the Cd1 group. Furthermore, the relative abundance of other known metal-resistant and detoxifying genera—*Herbaspirillum*, *Enterobacter*, *Pantoea*, *Rhodobacter*, and *Hydrogenophaga*—also increased significantly with increasing cadmium concentration (P<0.05). These compositional changes confirm that the microbial community of *Lemna minor* leaves not only tolerates cadmium stress but also actively responds to it through the selective enrichment of specific genera.

[0063] LEfSe analysis of bacterial community on leaves of *Lycoris radiata*

[0064] Differential bacterial comparison analysis was performed with an LDA score threshold of 4. The addition of cadmium altered the composition of bacterial communities at different levels (class, order, family, genus, and species) in the CK and Cd10 groups (Figure 6). At the family level, Cd10 was enriched relative to CK with Erwinaceae, Oxalobacteriaceae, and Enterobacteriaceae, while the CK group was enriched with Comamonadaceae and Sphingomonadaceae. At the genus level, the Cd10 group showed higher relative abundances of Pantoea, Herbaspirillum, and Enterobacter. These results are consistent with previous reports: in rice seeds, Enterobacteriaceae accounted for 15.48% of the endophytic community and was significantly negatively correlated with cadmium uptake; seed-derived endophytes (including Pantoea, Herbaspirillum, and Enterobacter) reduced cadmium accumulation in rice. In a study of duckweed rhizosphere responses to cadmium and microplastics, Comamonadaceae was the most abundant family in the control group.

[0065] The microbial community composition on the surface of *Lemna minor* leaves is closely related to host secretions and the microenvironment. Environmental heterogeneity is a major driver of speciation, and community remodeling under cadmium stress may be induced by multiple factors, including nutrient limitation caused by heavy metal accumulation. In summary, these results indicate that *Lemna minor* forms unique microbial communities under different cadmium concentrations.

[0066] Experiment 6: Prediction of bacterial function in duckweed leaves under different treatments

[0067] To infer the metabolic potential of leaf-associated bacterial communities under cadmium stress, this study used PICRUSt2 based on functional information from phylogenetic and known genome databases. Gene function analysis of the microbial community was performed using the KEGG and COG pathways based on 16S rRNA gene data (Figure 7). COG is a database for classifying homologous gene products, which can assess homologous genes by comparing protein sequences of certain organisms. The results showed that, compared with the CK group, several COG classification functions (such as predicted transporters, mevalonate pyrophosphate decarboxylase, and cephalosporin hydroxylase) were significantly downregulated in the Cd10 group (P<0.05). Conversely, cadmium exposure significantly enriched the abundance of genes encoding antitoxins isolated from F plasmids, the nitrate / nitrite transporter NarK, and components of the ABC-type Mla transport system (including MlaB containing the STAS domain, whose function is to maintain outer membrane lipid asymmetry) (P<0.05).

[0068] The KEGG database is a systematic database for investigating the metabolic pathways of gene products and their functions in living cells. Results showed that, compared to the control group (CK), both Cd1 and Cd10 treatments significantly increased the relative abundance of signal transduction, ion channel function, and pathways related to nucleotide and riboflavin metabolism (P<0.05); conversely, terpene skeletal biosynthesis pathways were significantly downregulated under cadmium stress (P<0.05). Notably, the abundance of universal transport pathways was higher in the Cd10 group than in the CK group, while the abundance of transport pathways was decreased in the Cd1 group. These functional changes suggest that *Leptochloa crus-galli*-associated bacteria may adapt to cadmium stress by remodeling their metabolic profile—particularly transport, signaling, and detoxification-related functions.

[0069] Example 2:

[0070] 1. Prepare bacterial suspensions separately:

[0071] The *Syntrophus* spp., *Enterobacter* spp., and *Pantotheca* spp. were purchased from the China Microbial Culture Collection Platform (Platform No.: Bio-59026), respectively (Platform No.: Bio-62157), respectively (Platform No.: Bio-60429).

[0072] Preparation of bacterial suspension: One loopful of *Streptococcus* spp. was inoculated into LB liquid medium (10.0 g tryptone, 5.0 g yeast extract, 10.0 g sodium chloride, 1000.0 mL distilled water, pH 7.0, autoclaved at 121℃ for 20 min). The suspension was then cultured in a shaker at 30℃ and 150 r / min for 48 h. After centrifugation at 8000 rpm for 10 min, the supernatant was removed, and the cells were washed with PBS buffer. Sterile water was then added to prepare a 10% suspension. 6 CFU / mL of *Streptococcus* suspension.

[0073] One loopful of Enterobacter spp. was inoculated into BPY liquid medium (5.0 g beef extract, 10.0 g peptone, 5.0 g yeast extract, 5.0 g glucose, 5.0 g NaCl, 1000.0 mL distilled water, pH 7.0, autoclaved at 121℃ for 20 min). The medium was then incubated at 30℃ and 150 r / min in a shaker for 48 h. Afterward, the medium was centrifuged at 8000 rpm for 10 min, the supernatant was removed, and the cells were washed with PBS buffer. Sterile water was then added to prepare a 10% concentration. 6 CFU / mL Enterobacter spp. suspension.

[0074] One loopful of *Pantheraea* bacteria was inoculated onto LB broth and cultured in a shaker at 30°C and 150 rpm for 48 h. The culture was then centrifuged at 8000 rpm for 10 min, the supernatant was removed, and the cells were washed with PBS buffer. Sterile water was then added to prepare a 10% concentration solution. 6 CFU / mL Pantotheca spp. suspension.

[0075] 2. Preparation of mixed bacterial suspension:

[0076] The above-prepared suspensions of *Streptococcus*, *Enterobacter*, and *Pantotheca* were mixed in a mass ratio of 2.5:2.5:1 to prepare a mixed bacterial suspension.

[0077] Take 1L of water from cadmium-contaminated water and place it in a hydroponic container. Mix duckweed and a mixed bacterial suspension at a mass ratio of 1:0.05. Then, at 25±1 ℃, with 16 h of light / 8 h of darkness and a light intensity of 40 μmol•m -2 •s -1 The samples were cultured under specific conditions and collected on day 7. The cadmium content was determined, with each measurement group repeated three times.

[0078] Water samples: Each time a sample is taken, a portion of the water is drawn up with a syringe, filtered through a membrane, acidified, and then measured using an inductively coupled plasma atomic emission spectrometer.

[0079] Biological Samples: At the end of the experiment, all *Lemna minor* samples were collected, thoroughly rinsed with deionized water, ground, and weighed to 2.0 g. The samples were placed in a 50 mL dry glass digestion tube, and 10 mL of concentrated nitric acid was added. The tube was incubated overnight. The digestion tube was then placed in a digestion furnace to accelerate digestion. The digest was heated to 70 °C within 15 minutes, held for 30 minutes, then heated to 90 °C within 30 minutes, and finally allowed to stand at 120 °C for 2 hours. The digestion was then removed from the funnel. After cooling to room temperature, the solution was diluted to 40 mL with ultrapure water, filtered through filter paper, and then measured using inductively coupled plasma atomic emission spectrometry (ICP-AES). The specific results are shown in Table 2.

[0080] Table 2:

[0081]

[0082] The results are shown in the table above. The cadmium content in the untreated water was 209.02 mg / L. After the combined use of duckweed and microbial inoculant (mixed bacterial suspension), the cadmium content in the water decreased to 0.23 mg / L. At this time, the cadmium content in duckweed was 1213.74 mg / L.

[0083] Example 3:

[0084] The preparation of the mixed bacterial suspension is the same as in Example 2.

[0085] Soil samples were collected from the experimental field to determine the content of the heavy metal cadmium.

[0086] Add duckweed and mixed bacterial suspension to the experimental field at a mass ratio of 1:0.05. Select rice seedlings with uniform growth and transplant them into the experimental field. Maintain a water layer of 3-6 cm throughout the entire growth period. Apply urea during the rice tillering stage, and plant the remaining rice seedlings according to existing techniques.

[0087] Determination of available cadmium content in soil: 5.0 g of soil sample was dispersed in 25 ml of 1% hydrochloric acid. The soil was rotated and shaken, and then the filtrate was obtained through a 0.45 μm filter membrane for determination of available Cd concentration, which was then measured using inductively coupled plasma atomic emission spectrometry (ICP-AES).

[0088] Cadmium content determination in rice: According to the national standard GB 5009.15-2023 "National Food Safety Standard - Determination of Cadmium in Food", after rice is harvested, the husk is removed, the rice is pulverized into a uniform powder, digested with nitric acid by microwave, and determined by inductively coupled plasma mass spectrometry.

[0089] Tests showed that rice grown in experimental fields with mild soil cadmium pollution (<0.7 mg / kg) achieved a cadmium content of 0.01-0.078 mg / kg in rice by applying a Zi Ping-compound microbial agent, which is far below the national limit of 0.2 mg / kg.

[0090] Those skilled in the art should understand that the methods described in this invention are not limited to the embodiments described in the specific implementation details. The above detailed description is merely for illustrative purposes and is not intended to limit the invention. Other implementation methods derived by those skilled in the art based on the technical solutions of this invention also fall within the scope of this invention's technical innovation. The scope of protection of this invention is defined by the claims and their equivalents.

Claims

1. The application of duckweed combined with microorganisms in the remediation of cadmium-contaminated water, characterized in that, The mass ratio of duckweed to microorganisms is 1:0.01-0.1; the microorganisms include *Streptococcus*, *Enterobacter*, and *Pantospira*; the mass ratio of *Streptococcus*, *Enterobacter*, and *Pantospira* is 2.2-2.8:2.2-2.8:1; the specific application method includes the following steps: first, prepare *Streptococcus*, *Enterobacter*, and *Pantospira* suspensions separately; then mix the three suspensions in a certain proportion to obtain a mixed suspension; next, add the mixed suspension and duckweed in a certain proportion to the cadmium-polluted water body to be remediated, and duckweed and microorganisms form a "duckweed-functional microbial community" symbiotic system; the microorganisms then transport cadmium to the leaves through adsorption, transformation, leaf surface sedimentation, or transpiration; finally, the duckweed is centrally treated to achieve the remediation of cadmium-polluted water body.

2. The application of *Lysimachia christinae* combined with microorganisms in reducing cadmium content in rice grains from cadmium-polluted paddy fields, characterized in that... The mass ratio of duckweed to microorganisms is 1:0.01-0.1; the microorganisms include *Streptococcus*, *Enterobacter*, and *Pantospira*; the mass ratio of *Streptococcus*, *Enterobacter*, and *Pantospira* is 2.2-2.8:2.2-2.8:1; the specific application method includes the following steps: first, prepare *Streptococcus*, *Enterobacter*, and *Pantospira* suspensions separately; then mix the three suspensions in a certain proportion to obtain a mixed suspension; next, add the mixed suspension and duckweed in a certain proportion to the cadmium-polluted paddy field to be remediated, and duckweed and microorganisms form a "duckweed-functional microbial community" symbiotic system; the microorganisms then transport cadmium to the leaves through adsorption, transformation, leaf surface sedimentation, or transpiration; finally, the duckweed is centrally treated to reduce the cadmium content of rice grains.

3. The application as described in claim 1 or 2, characterized in that: The mass ratio of *Streptococcus*, *Enterobacter*, and *Pantotheca* was 2.5:2.5:

1.

4. The application as described in claim 1 or 2, characterized in that: The mass ratio of duckweed to microorganisms was 1:0.

05.

5. The application as described in claim 1, characterized in that: The cadmium content in cadmium-polluted water bodies should not exceed 2 μM.

6. The application as described in claim 1, characterized in that: After remediation, the concentration of cadmium in the water was <0.2276 mg / kg.

7. The application as described in claim 2, characterized in that: The cadmium content in cadmium-contaminated paddy fields is no higher than 2 μM.

8. The application as described in claim 2, characterized in that: After application, the cadmium content in rice grains was <0.20 mg / kg.

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