Method for purifying chromium-containing water body through combination of arbuscular mycorrhizal fungi and reed and application

Abstract

The invention provides a method for purifying a chromium-containing water body through combination of arbuscular mycorrhizal fungi and reeds and application, and relates to the technical field of environmental engineering and ecological restoration. The problems that an existing physical and chemical method is high in cost and prone to secondary pollution, and single plant remediation is low in efficiency, long in period and the like are solved. The method comprises the following steps: firstly, constructing an arbuscular mycorrhizal fungus-reed combination in a sterile solid substrate; then, the constructed arbuscular mycorrhizal fungi-reed combination is transplanted into a chromium-containing water body for purification treatment; the arbuscular mycorrhizal fungi are root interior root cyst fungi, and the reeds are reed seedlings. The method is applied to the chromium-containing water body, the operation process of the method is clear, parameters are controllable, and the synergistic purification effect of '1 + 1gt; 2' can be stably achieved. The used plants and fungicide materials are wide in source, complex equipment is not needed in the culture and purification process, the cost is low, secondary pollutants are not introduced in the whole remediation process, the environmental compatibility is excellent, and the method has good application and popularization prospects.

Description

Technical Field

[0001] This invention relates to the field of environmental engineering and ecological restoration technology, specifically to a combined arbuscular mycorrhizal fungus and reed purification method. Methods and applications for treating chromium-containing water bodies. Background Technology

[0002] Chromium (Cr) is a typical heavy metal pollutant, especially its hexavalent form (Cr(VI)), which is highly toxic, highly mobile, and carcinogenic, posing a serious threat to aquatic ecosystems and human health. Currently, the treatment of chromium-containing wastewater mainly relies on physicochemical methods such as chemical precipitation, ion exchange, and membrane separation. While these methods can effectively remove chromium, they generally have limitations such as high treatment costs, complex operations, and the potential for secondary pollution (e.g., chromium-containing sludge), making them unsuitable for the ecological restoration of large-scale, low-to-medium concentration polluted water bodies (such as mine leachate and polluted rivers and lakes).

[0003] In recent years, phytoremediation technology has been regarded as a promising alternative or supplementary solution for heavy metal pollution control due to its advantages such as low cost, environmental friendliness, and ecological harmony. (Reed) Phragmites australis Phytoremediation, as a common emergent wetland plant, is characterized by its large biomass, strong adaptability, and well-developed root system. It also exhibits a certain tolerance and accumulation capacity for various heavy metals and is widely used in constructed wetland treatment systems. However, phytoremediation alone often faces bottlenecks such as limited remediation efficiency, inhibited plant growth under heavy metal stress, and long remediation cycles.

[0004] Arbuscular mycorrhizal fungi (AMF) are soil fungi that can form mutually beneficial symbiotic relationships with the roots of most terrestrial plants. Studies have shown that AMFs not only help host plants expand their nutrient uptake range through their extensive extra-rhizomatous hyphal networks, but also enhance the host plant's tolerance to heavy metal stress through various mechanisms (such as direct hyphal adsorption, mycorrhizal interface fixation, induction of plant antioxidant systems, and alteration of the rhizosphere microenvironment), thereby improving the plant's survival ability and remediation efficiency in polluted environments. Combining AMFs with hyperaccumulating or tolerant plants to construct a "plant-microbe" co-remediation system has become a research hotspot for improving the remediation effect of heavy metal pollution.

[0005] However, applying the AMF-plant symbiotic system to the purification of chromium pollution in water bodies still faces many challenges: First, AMF is an obligate, biotrophic microorganism, and its infection efficiency and the establishment of the symbiosis strongly depend on specific host plant species, fungal species, and environmental conditions (such as water pH, nutrient status, and pollutant concentration). Not all plant-AMF combinations can stably establish an efficient symbiotic relationship in a hydroponic environment with heavy metals. Second, existing research mainly focuses on soil remediation. For dynamic aquatic environments (such as changes in flow rate and dissolved oxygen), there is a lack of systematic methodological guidance and process optimization to achieve efficient and stable colonization of AMF in reed roots and maximize its synergistic chromium purification efficiency. Finally, many technologies remain at the stage of laboratory mechanism research, and there is still a long way to go before forming replicable and controllable engineering application solutions.

[0006] Publication number CN114956366A, invention titled "A method and application of using arbuscular mycorrhizal fungi to enhance phytoremediation of chromium-contaminated soil," discloses the use of *Morchella mosierifolia* (… Funneliformis mosseae ) and associated mineral Sedum ( Sedum plumbizincicola This invention describes a method for remediating chromium-contaminated soil using a combination of mycorrhizal fungi (AMF) and emergent plant symbionts. The invention demonstrates that AMF can improve the efficiency of chromium extraction by plants. However, this method targets soil media, and the application methods of the fungal agent (such as mixing with soil) and the symbiotic cultivation conditions differ fundamentally from hydroponic environments. The patent does not address how to construct and apply an AMF-emergent plant symbiont in aquatic environments, particularly failing to solve key engineering and technical issues such as maintaining mycorrhizal activity in flowing or static water and optimizing process parameters (such as strain selection, inoculation method, and control of water physicochemical conditions) for the synergistic purification of chromium (VI) by fungi and plants.

[0007] Therefore, developing a patented technology that can efficiently construct an arbuscular mycorrhizal fungus-reed symbiosis and clarify the specific conditions and methods for its application in purifying chromium-containing water is of great practical significance for promoting the practical engineering application of plant-microbe joint remediation technology. Summary of the Invention

[0008] This invention addresses the problems of existing chromium-contaminated water remediation technologies, such as the high cost and potential for secondary pollution associated with physicochemical methods, the limited efficiency and long cycle of single-plant remediation technologies, and the fact that existing plant-microbe co-remediation technologies are mostly designed for soil environments and lack a systematic approach suitable for water purification that can stably construct efficient symbionts. Therefore, this invention proposes a new... A method and application of arbuscular mycorrhizal fungi combined with reeds to purify chromium-containing water bodies, which can significantly improve the purification efficiency of chromium (especially hexavalent chromium) polluted water bodies.

[0009] The technical solution adopted by the present invention to solve the above problems is as follows: This invention proposes a method for purifying chromium-containing water using arbuscular mycorrhizal fungi and reeds in combination. The method employs a phased implementation strategy, comprising two sequential phases: Phase 1: Construction of arbuscular mycorrhizal fungi-reed complex in a sterile solid matrix; The second stage: The constructed arbuscular mycorrhizal fungi-reed complex was transplanted into chromium-containing water for purification treatment. The arbuscular mycorrhizal fungus is *Rhizocystis ulmoides*, and the reed is a reed seedling.

[0010] Furthermore, in the first stage of constructing the arbuscular mycorrhizal fungi-reed complex, a simultaneous sowing and inoculation method is adopted, in which reed seeds and root endomycorrhizal fungi are co-cultured in a sterilized substrate, with 30-50 spores inoculated per kilogram of substrate, and cultured until the reed seedlings are 15-25 cm tall, have 4-6 fully expanded leaves, and the root system forms an effective mycorrhizal symbiosis.

[0011] Furthermore, the construction of the arbuscular mycorrhizal fungi-reed complex is specifically as follows: sterilized seedling substrate is evenly mixed with root endomycorrhizal fungi agent, then reed seeds are sown, and cultured under suitable conditions for 4-6 weeks until the reed seedling roots form an effective and stable mycorrhizal symbiosis, thus completing the construction of the complex.

[0012] Furthermore, the chromium-containing water bodies mentioned in the second phase are hexavalent chromium-polluted water bodies.

[0013] Furthermore, the second stage of purification treatment is as follows: the healthy arbuscular mycorrhizal fungi-reed synthesis seedlings cultivated in the first stage are removed from the original substrate and their roots are washed. They are then planted in a hydroponic device, and water containing hexavalent chromium is added to the device for purification cultivation under natural or artificial light conditions.

[0014] Furthermore, in the water body containing hexavalent chromium, the initial concentration of hexavalent chromium is 10-100 mg / L, the pH value of the water body is 5.5-7.5, and the culture temperature is 20-30℃.

[0015] This invention also proposes an application of the above method in the combined purification of chromium-containing water by arbuscular mycorrhizal fungi and reeds.

[0016] Furthermore, the chromium-containing water body is a hexavalent chromium-polluted water body with an initial concentration of 10-100 mg / L, a pH value of 5.5-7.5, and a culture temperature of 20-30℃.

[0017] The beneficial effects of this invention are: 1. Two-stage approach to ensure efficient and stable symbiosis: This invention employs a two-stage method: first, solid substrate cultivation, then water transplantation and purification. In the first stage, simultaneous inoculation via sowing in a controlled, sterile substrate provides an ideal environment for the establishment of symbiosis between reed seedlings and root-borne mycorrhizal fungi, ensuring efficient and stable colonization of the mycorrhizae and the formation of a robust complex. In the second stage, this complex is then transplanted into the polluted water body, allowing it to cope with chromium stress based on its already strong symbiotic function, significantly improving the success rate and stability of the remediation system.

[0018] 2. Synergistic Effect, Overcoming the Bottleneck of Single-Remediation: The "AMF-Reed" consortium constructed in this invention exhibits a significant synergistic purification effect. On the one hand, AMF directly adsorbs and immobilizes some chromium ions through its extensive hyphal network and improves the rhizosphere microenvironment of reeds, indirectly promoting reed growth. On the other hand, AMF symbiosis significantly enhances the physiological tolerance of reeds to chromium stress, increasing their biomass and chromium accumulation capacity. This synergistic effect makes the consortium's removal efficiency, stability, and speed of hexavalent chromium in water significantly superior to that of single-reed reed remediation without AMF inoculation.

[0019] 3. Clear methodology, strong operability and practicality: This invention provides a complete technical solution and specific parameter range, from the construction of the consortium (including specific strains, inoculum size, plant state, and culture period) to application conditions (chromium concentration, pH, and temperature). The method has clear steps and requires conventional materials, allowing for precise verification on a laboratory scale and easy scaling up for engineering remediation scenarios such as constructed wetlands and ecological floating islands, effectively transforming laboratory mechanism research into practical application technology.

[0020] 4. Environmental friendliness and cost advantages: This invention is entirely based on the principle of bioremediation, without the addition of chemical agents, posing no risk of secondary pollution, and exhibiting excellent environmental compatibility. By optimizing the inoculation strategy, efficient root colonization can be achieved with a relatively small amount of AMF spores, reducing the cost of inoculants. Furthermore, the use of reeds, a common wetland plant, ensures readily available raw materials, giving this technology overall the potential for low-cost and sustainable application.

[0021] 5. Provides an innovative approach to solving chromium pollution in water bodies: Compared with existing AMF-plant co-remediation technologies that mainly focus on soil remediation, this invention is specifically designed for aquatic environments, clarifying the key technical conditions for successfully establishing and applying an AMF-plant symbiotic system in this medium, and providing a practical solution for expanding the application scope of plant-microbe co-remediation technology to the field of water pollution control. Attached Figure Description

[0022] Figure 1 A schematic diagram of mycorrhizalized reed culture; Figure 2This is a schematic diagram of the experiment on constructing the arbuscular mycorrhizal fungus-reed complex using the method of the present invention and its application in chromium-containing water (10 mg / L); Figure 2 (a) shows the culture status of reed assemblages inoculated with Rhizocystis nematodes in chromium-containing water, and (b) shows the culture status of uninoculated reeds in the same chromium-containing water.

[0023] Figure 3 This is a microscopic observation of mycorrhizal infection of reed roots in the method of this invention; Figure 3 (a) is a stained section of reed roots inoculated with Rhizocystis suis and subjected to chromium stress (showing typical hyphae, vesicles and arbuscular structures). (b) is a stained section of roots from the uninoculated control group (without typical AMF structure). Detailed Implementation

[0024] This embodiment proposes a method for purifying chromium-containing water bodies using arbuscular mycorrhizal fungi (AMM) and reeds in a combined manner. The method employs a phased implementation strategy: First, an AMM-reed complex is constructed under controlled solid substrate conditions; the AMM is *Rhizocarpus oryzae*, and the reeds are reed seedlings. During the construction of the AMM-reed complex, a simultaneous sowing and inoculation method is used. *Rhizocarpus oryzae* spores are uniformly mixed with sterilized seedling substrate, and then reed seeds are sown. The spore inoculation rate is 30-50 spores / kg substrate. When the reed seedlings reach a height of 15-25 cm and have 4-6 fully expanded leaves, robust mycorrhizalized seedlings are obtained.

[0025] The construction of the arbuscular mycorrhizal fungi-reed complex is specifically as follows: sterilized seedling substrate is evenly mixed with root endomycorrhizal fungi agent, then reed seeds are sown, and cultured under suitable conditions for 4-6 weeks until the reed seedling roots form an effective and stable mycorrhizal symbiosis, thus completing the construction of the complex.

[0026] Then, the robust complex that has formed a stable symbiotic relationship is transplanted and applied to the chromium-containing water body to be purified for purification treatment; The chromium-containing water to be purified is hexavalent chromium-polluted water.

[0027] The specific steps for purifying hexavalent chromium-polluted water are as follows: the constructed arbuscular mycorrhizal fungi-reed complex seedlings are removed from the substrate and their roots are washed. They are then planted in a hydroponic or simulated wetland device. Subsequently, water containing hexavalent chromium is added to the device, and the water is cultured and purified under natural or artificial light conditions.

[0028] The initial concentration of hexavalent chromium in the water to be treated is 10-100 mg / L, the pH value of the water is 5.5-7.5, and the culture temperature is 20-30℃.

[0029] This embodiment also proposes an application of the above method in the combined purification of chromium-containing water by arbuscular mycorrhizal fungi and reeds. The chromium-containing water is hexavalent chromium-polluted water with an initial concentration of 10-100 mg / L, a pH of 5.5-7.5, and a culture temperature of 20-30℃. The technical solution of the present invention will be further described below with reference to embodiments and accompanying drawings.

[0030] Example 1: In 2025, reed cultivation was carried out at the Key Laboratory of Cold Region Ecological Restoration and Resource Utilization of Heilongjiang University. I. Experimental Preparation 1. Test plants: Select reed seeds that are uniform in size and plump ( Phragmites australis ).

[0031] 2. Test inoculum: Rhizocystis ulmoides ( Rhizophagus irregularis (abbreviated as Ri). The spore content of the experimental fungal agent was approximately 35-50 spores / g (preserved and provided by the Restoration Ecology Research Laboratory of Heilongjiang University).

[0032] 3. Mycorrhizal cultivation substrate: A mixture of river sand and peat moss (volume ratio 3:1) sterilized by high-temperature steam (121°C, 2 hours) was used as the carrier for constructing mycorrhizal complexes.

[0033] 4. Preparation of polluted water: Dissolve potassium dichromate (K2Cr2O7) in deionized water to prepare simulated polluted water with an initial hexavalent chromium concentration of 10 mg / L. Adjust the pH to 6.5 ± 0.2 with 0.1 mol / L NaOH or HCl solution.

[0034] II. First Stage: Construction of the Consortium (Pre-culture / Mycorrhizalization Stage) 1. Fill the sterilized substrate into a plastic seedling pot with an upper diameter of 15 cm.

[0035] 2. Inoculation Treatment Group (AM): The sowing-simultaneous inoculation method was adopted. The root endophytic fungicide was mixed evenly with the sterilized substrate at a ratio of 10 g / kg (fungicide / substrate), and then the reed seeds were sown in the substrate.

[0036] 3. Control group (CK): A control group was established where only reed seeds were sown in sterilized substrate without inoculation with any inoculum.

[0037] 4. All treatments were placed in an artificial climate incubator and irrigated with Hoagland nutrient solution at half intensity. The incubation conditions were: day / night temperature 25 / 20°C, photoperiod 14h / 10h, and light intensity 300 μmol·m⁻¹. - ²·s - ¹. The pre-culture period is 6 weeks to ensure the stable establishment of the mycorrhizal symbiosis.

[0038] III. Second Stage: Water Purification Experiment (Transplantation and Application Stage) 1. After the pre-culture is completed, carefully remove the plant and gently rinse the roots with deionized water to remove the substrate attached to them.

[0039] 2. Transplant the reed seedlings into a hydroponic container containing 2.0 L of the simulated chromium-contaminated water (e.g., ...). Figure 1 (As shown). Five biological replicates were set up for each treatment.

[0040] 3. The purification experiment was conducted in the same culture chamber environment for 21 days. During the experiment, the deionized water lost through evaporation was replenished daily to maintain a constant volume, and the pH was monitored and adjusted to 6.5 weekly.

[0041] IV. Measurement and Analysis Methods The following measurements were performed after the experiment: 1. Chromium residue in water: Water samples were collected, and the concentration of residual hexavalent chromium was determined by inductively coupled plasma mass spectrometry (ICP-MS) to calculate the chromium removal rate.

[0042] 2. Plant biomass and chromium accumulation: Reeds were harvested and separated into aboveground parts and roots, and their dry weight was measured. After digestion with nitric acid and perchloric acid, the chromium content of the plant samples was determined by inductively coupled plasma mass spectrometry (ICP-MS).

[0043] 3. Mycorrhizal colonization rate: Fresh fine roots were taken, and slides were prepared using the trypan blue staining method. The mycorrhizal colonization rate was observed and counted under a microscope.

[0044] The colonization rate and infection intensity of arbuscular mycorrhizal fungi on the roots of reed seedlings were detected, and the results are shown in Table 1.

[0045] Table 1.

[0046] Note: The purification effect of different treatments on chromium-containing water (10 mg / L) and the response of reed growth. Compared to the uninoculated control group, reeds inoculated with AMF showed an approximately 31.6% higher chromium removal rate from the water, and a significant increase in plant biomass (aboveground and roots). Simultaneously, the aboveground chromium content of the inoculated reeds was lower, while the root chromium content remained at a higher level, indicating that AMF may mitigate the translocation and toxicity of chromium to the aboveground parts by fixing it in the roots or altering its morphology, thereby enhancing the overall plant tolerance and remediation efficiency. High mycorrhizal colonization rate confirmed the successful establishment of AMF and the reed root system.

[0047] To further investigate the synergistic effect of arbuscular mycorrhizal fungi (ARF) on reed roots, the colonization rate, infection intensity, and Cr removal rate of ARF fungi on reed roots were examined at different Cr concentrations. Table 2 shows that the mycorrhizal reed symbiont provided by this invention can effectively purify water bodies and maintain a high mycorrhizal colonization rate within a Cr concentration range of 10-100 mg / L. Although the purification efficiency, plant biomass, and colonization rate decreased with increasing Cr concentration, a removal rate of over 69% and a colonization rate of nearly 50% were still maintained at concentrations as high as 100 mg / L, demonstrating that this method has broad concentration adaptability and good stress tolerance.

[0048] The key results after 21 days of purification experiment are shown in Table 2.

[0049] Table 2.

[0050] Note: Inoculation refers to grafting with *Rhizocystis suis* (a fungus that inoculates the root). Rhizophagus irregularis Collection number: CGMCCNo.10607.

[0051] Infection intensity: + (0-10); ++ (10-20); +++ (20-30); ++++ (30-40); +++++ (40-50).

[0052] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent substitutions, and improvements made to the above embodiments without departing from the scope of the present invention, based on the technical essence of the present invention and within the spirit and principles of the present invention, shall still fall within the protection scope of the present invention.

Claims

1. A method for purifying chromium-containing water by combining arbuscular mycorrhizal fungi and reeds, characterized in that, The method comprises two sequentially connected stages: Phase 1: Construction of arbuscular mycorrhizal fungi-reed complex in a sterile solid matrix; The second stage: The constructed arbuscular mycorrhizal fungi-reed complex was transplanted into chromium-containing water for purification treatment. The arbuscular mycorrhizal fungus is *Rhizocystis ulmoides*, and the reed is a reed seedling.

2. The method for purifying chromium-containing water by combining arbuscular mycorrhizal fungi and reeds according to claim 1, characterized in that, In the first stage, when constructing the arbuscular mycorrhizal fungus-reed complex, the sowing and inoculation method is adopted. Reed seeds and root endomycorrhizal fungi are co-cultured in a sterilized substrate, with 30-50 spores inoculated per kilogram of substrate. The seedlings are cultured until they reach a height of 15-25 cm, have 4-6 fully expanded leaves, and the root system forms an effective mycorrhizal symbiosis.

3. The method for purifying chromium-containing water by combining arbuscular mycorrhizal fungi and reeds according to claim 2, characterized in that, The construction of the arbuscular mycorrhizal fungi-reed complex is specifically as follows: sterilized seedling substrate is evenly mixed with root endomycorrhizal fungi agent, then reed seeds are sown, and cultured under suitable conditions for 4-6 weeks until the reed seedling roots form an effective and stable mycorrhizal symbiosis, thus completing the construction of the complex.

4. The method for purifying chromium-containing water by combining arbuscular mycorrhizal fungi and reeds according to claim 1, characterized in that, The chromium-containing water bodies mentioned in the second stage are water bodies polluted by hexavalent chromium.

5. The method for purifying chromium-containing water by combining arbuscular mycorrhizal fungi and reeds according to claim 4, characterized in that, The second stage of purification treatment is as follows: After the healthy arbuscular mycorrhizal fungi-reed synthesis seedlings cultivated in the first stage are taken out of the original substrate and their roots are washed, they are planted in a hydroponic device. Then, water containing hexavalent chromium is added to the device and purified under natural or artificial light conditions.

6. The method for purifying chromium-containing water by combining arbuscular mycorrhizal fungi and reeds according to claim 5, characterized in that, The water body containing hexavalent chromium has a pH value of 5.5-7.5, an initial concentration of hexavalent chromium of 10-100 mg / L, and a culture temperature of 20-30℃.

7. The application of the method according to any one of claims 1 to 6 in the combined purification of chromium-containing water by arbuscular mycorrhizal fungi and reeds.

8. The application of the arbuscular mycorrhizal fungi and reeds in the combined purification of chromium-containing water according to claim 7, characterized in that, The chromium-containing water body is a hexavalent chromium-polluted water body with a pH value of 5.5-7.5, an initial concentration of hexavalent chromium of 10-100 mg / L, and a culture temperature of 20-30℃.

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