Fungus ball composite material for enhancing denitrification of micro-polluted water and preparation method thereof

Abstract

The application discloses a preparation method of mycelial pellet composite material for enhanced denitrification of micro-polluted water, and comprises the following steps: screening and obtaining a bacterial strain and a fungal strain, and preparing a bacterial suspension; culturing the fungal strain to prepare mycelial pellets; culturing the bacterial suspension in a mycelial pellet culture medium to prepare DMP; modifying biochar by KMnO4 to prepare modified biochar, and preparing a modified biochar suspension; adding the modified biochar suspension and a chlorella suspension into the DMP, and oscillating and culturing to prepare the mycelial pellet composite material. The mycelial pellet composite material for enhanced denitrification of micro-polluted water prepared by the application has the characteristics of structural stability, high electron transfer efficiency and strong adaptability.

Description

Technical Field

[0001] This invention belongs to the field of water treatment functional materials and biological denitrification technology, specifically relating to mycelial ball composite materials for enhanced denitrification of slightly polluted water, and also to a method for preparing mycelial ball composite materials for enhanced denitrification of slightly polluted water. Background Technology

[0002] Slightly polluted water typically refers to surface water, landscape water, or water bodies affected by domestic sewage or agricultural non-point source pollution, which have a relatively low level of pollution but contain certain concentrations of nitrogen, phosphorus, and organic matter. The continuous accumulation of nitrogen, especially nitrate nitrogen and total nitrogen, in such water bodies easily leads to eutrophication, abnormal algal growth, and ecological degradation. Therefore, developing efficient and stable denitrification technologies suitable for slightly polluted water is of great significance. Slightly polluted water generally suffers from insufficient organic carbon sources, fluctuating nitrogen concentrations, easy loss of microorganisms, and insufficient system stability. Traditional biological denitrification methods, such as suspended activated sludge processes or single-functional bacteria methods, have limited denitrification efficiency under low carbon-to-nitrogen ratio conditions.

[0003] Mycelial balls formed by filamentous fungi can serve as microbial immobilization carriers. The symbiotic system constructed by these mycelial balls and denitrifying bacteria can improve nitrate removal rates, promote extracellular polymer secretion, and enhance electron transport activity under low C / N ratio conditions. Meanwhile, biochar, due to its large specific surface area, abundant pore structure, and surface functional groups, is used to enhance biological nitrogen removal processes. Biochar not only serves as a microbial attachment carrier but also enhances denitrification by promoting electron transport, improving mass transfer conditions, and buffering environmental fluctuations.

[0004] Existing biological nitrogen removal technologies mainly focus on single mycelial ball immobilized denitrification systems, simple biochar-enhanced systems, or general bacterial-algae immobilization systems. For nitrogen removal needs in slightly polluted water, especially under low C / N ratio conditions, existing technologies still have the following shortcomings: First, while single mycelial ball immobilization systems can improve microbial retention, the efficiency of external electron donors and transfer remains limited, and long-term operational stability needs improvement. Second, although ordinary biochar has a certain enhancing effect, its surface activity, conductivity, and ability to promote microbial attachment and electron transfer remain limited without targeted modification. Third, existing bacterial-algae immobilization systems mostly rely on gel carrier encapsulation, and composite nitrogen removal materials synergistically constructed with fungal mycelial ball carriers, aerobic denitrifying bacteria, and modified biochar are rare, resulting in insufficient adaptability for the efficient removal of nitrate and total nitrogen in slightly polluted water. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing mycelial ball composite materials for enhanced denitrification of slightly polluted water.

[0006] Another objective of this invention is to provide a mycelial ball composite material for enhanced denitrification of slightly polluted water, which features structural stability, high electron transfer efficiency, and strong adaptability.

[0007] The technical solution adopted in this invention is a method for preparing mycelial ball composite materials for enhanced denitrification of slightly polluted water. The method for preparing mycelial ball composite materials for enhanced denitrification of slightly polluted water includes the following steps:

[0008] Bacterial and fungal strains were screened to obtain bacterial and fungal strains, and bacterial suspensions were prepared. Culture fungi to make mycelial balls; DMP was prepared by inoculating bacterial suspensions into mycelial ball culture medium; Modified biochar was prepared by modifying biochar with KMnO4, and a modified biochar suspension was prepared. Modified biochar suspension and Chlorella suspension were added to DMP and cultured by shaking to obtain mycelial ball composite material.

[0009] The invention is further characterized in that, The bacterial strain was *Bacillus pallida*, and the fungal strain was *Aspergillus*.

[0010] Bacterial and fungal strains were sampled and screened from water bodies, enriched in nitrate medium, and obtained by gradient dilution and coating.

[0011] The bacteria were confirmed through denitrification performance testing. The bacterial suspension was prepared by taking 15-20 ml of aerobic denitrification liquid culture medium containing bacterial spp. that had been incubated for 36-48 h, centrifuging at 5000-6000 r / min for 5-10 min, and discarding the supernatant to obtain the bacterial suspension.

[0012] Mycelial balls were prepared by culturing fungi in a medium containing NH4Cl, glucose, KH2PO4, and MgSO4·7H2O at 25-30℃ and 170-180 r / min.

[0013] DMP preparation involves adding 0.5-1.0 ml of bacterial suspension with OD600=1.0 to the mycelial ball culture medium and culturing at 25-30℃ and 170-180 r / min for 36-48 h.

[0014] The biochar was made from walnut shell biochar, which was ground through a 100-mesh sieve, treated with 2% KMnO4 solution at 90-100℃ for 20-24 hours, washed until neutral, and dried at 60-70℃ for 10-12 hours to obtain modified biochar.

[0015] The modified biochar suspension is prepared by dissolving 1.5-2.0g of modified biochar in 15-20ml of ultrapure water, sonicating for 10-15min, and letting stand for 3-4h.

[0016] The combined culture conditions are 25-30℃, 170-180r / min shaking culture for 36-48h, the amount of Chlorella suspension added is 4.5-5ml with OD600=1.0, and the amount of modified biochar suspension added is 15-20ml.

[0017] After the composite material is prepared, wash it with ultrapure water 2-3 times.

[0018] Another technical solution adopted in this invention is a mycelial ball composite material for enhanced denitrification of slightly polluted water, which is prepared by the above-mentioned preparation method of the mycelial ball composite material for enhanced denitrification of slightly polluted water.

[0019] The beneficial effects of this invention are: The mycelial ball composite material prepared by this invention for enhanced denitrification of slightly polluted water can significantly improve the nitrogen removal efficiency in slightly polluted water, and is especially suitable for enhanced denitrification under low carbon-nitrogen ratio conditions. Using KMnO4 to modify walnut shell biochar can improve the specific surface area, pore structure, surface functional groups and electrochemical activity of the material, thereby enhancing the adhesion, growth and electron transfer capabilities of microorganisms and improving the activity of functional groups. Introducing Chlorella can improve the microenvironment of the system and provide some carbon source, thereby further enhancing the overall denitrification performance and the structural stability of the mycelium ball composite material; It can improve ETSA (Electron Transport System Activity) and promote the denitrification process; It solves the problems of low denitrification efficiency, easy loss of microorganisms, insufficient material structure stability, limited electron transfer efficiency and poor operation stability of traditional biological denitrification systems under slightly polluted water conditions, especially under low carbon-nitrogen ratio conditions. With abundant raw material sources and feasible preparation methods, it has good engineering application value and promotion prospects. Attached Figure Description

[0020] Figure 1 This is a flowchart illustrating the preparation process of the mycelial ball composite material for enhanced denitrification of slightly polluted water according to the present invention. Figure 2 This describes the nitrate removal performance of the mycelium ball composite material of the present invention in simulated water. Figure 3 This describes the total nitrogen removal performance of the mycelium ball composite material of this invention in simulated water. Figure 4 This illustrates the changes in electron transport activity of the mycelium ball composite material of the present invention in simulated water. Figure 5 This is an example of the nitrate removal performance of the mycelium ball composite material of the present invention in actual water bodies; Figure 6 This describes the total nitrogen removal performance of the mycelium ball composite material of this invention in actual water bodies; Figure 7 This describes the changes in electron transfer activity of the mycelium ball composite material of the present invention in actual water bodies. Detailed Implementation

[0021] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0022] The preparation method of the mycelial ball composite material for enhanced denitrification of slightly polluted water according to the present invention is as follows: Figure 1 As shown, the specific steps are as follows: 1) Samples were taken from the landscape water body, and non-nitrate-utilizing bacteria were eliminated using nitrate-enriched culture medium, allowing denitrifying bacteria to grow. 9-10 ml of sample was added to 100-150 ml of nitrate-enriched culture medium (components shown in Table 1 below). The medium was incubated at 25-30℃ on a shaker at 170-180 rpm for 36-48 hours. After two repeated enrichment cultures, the resulting bacterial solution was serially diluted (10⁻⁶ ppm). -4 -10 -7 After being spread onto a culture medium, the culture medium was placed in a constant temperature incubator for 36-48 hours. Two different colonies grew on the culture medium plate: bacterial colonies and fungal colonies.

[0023] Bacterial colonies are small, smooth, moist, viscous, and have regular edges. Fungal colonies are larger, appearing as dry, fluffy particles.

[0024] Table 1 Enrichment Culture Media

[0025] 2) Using an inoculation loop, a suitable amount of bacterial cells was picked from the culture medium plate and added to sterilized aerobic denitrification liquid medium. The composition of the aerobic denitrification performance test medium is shown in Table 2 below. The medium was incubated at 25-30℃ and 170-180 r / min on a shaker, and the nitrate content was measured at 0 h and 48 h to determine whether denitrification capability was present. The bacterial strain was identified. Ochrobactrum (Aphthous bacillus); Table 2 Aerobic Denitrification Liquid Culture Medium

[0026] Preparation of *Alanobacterium* suspension: Take 15-20 ml of aerobic denitrification liquid culture medium containing *Alanobacterium* that has been cultured for 36-48 h, centrifuge at 5000-6000 r / min for 5-10 min, discard the supernatant to obtain *Alanobacterium* suspension.

[0027] 3) Using an inoculation loop, pick an appropriate amount of fungal cells from the culture medium plate and identify the fungal strain Aspergillus. Add an appropriate amount of fungal spores to the fungal mycelium ball culture medium. The composition of the fungal mycelium ball culture medium is shown in Table 3. Take 15-20 ml of the fungal mycelium ball liquid culture medium containing Aspergillus fungus that has been cultured for 36-48 h, centrifuge at 5000-6000 r / min for 5-10 min, discard the supernatant, and you will get the Aspergillus fungal suspension.

[0028] Add 0.5-1.0 mL of Aspergillus fungal suspension to the fungal mycelial ball culture medium and culture it in a shaker at 25-30℃ and 170-180 r / min to obtain Aspergillus mycelial balls.

[0029] Table 3 Fungal mycelial ball culture medium

[0030] 4) Add 0.5-1.0 ml (OD600=1.0) of *Bacillus cereus* suspension to the culture medium in which *Aspergillus* mycelial balls have been prepared, and incubate in a shaker at 170-180 r / min and 25-30℃ for 36-48 h to obtain DMP (*Bacillus cereus* + *Aspergillus* mycelial balls).

[0031] 5) The raw material for biochar was walnut shells. Walnut shell biochar was purchased, ground, passed through a 100-mesh sieve, and dried. Oxidative modification of the biochar involved treating it with a 2% KMnO4 solution under magnetic stirring (90-100℃) for 20-24 hours. After treatment, the biochar was filtered using a vacuum filter, and the resulting biochar was washed with ultrapure water until the washings were neutral. It was then dried in an oven at 60-70℃ for 10-12 hours. The obtained sample was labeled HTBC. KMnO4 (Potassium permanganate modified walnut shell biochar).

[0032] 6) Add 1.5-2.0g of the modified biochar powder to 15-20ml of ultrapure water and sonicate for 10-15min, then let stand for 3-4h to obtain a modified biochar suspension. 7) Add 4.5-5 ml of modified biochar suspension to DMP medium, and simultaneously add 4.5-5 ml of Chlorella suspension (OD600=1.0). Then, incubate in a shaker at 170-180 r / min and 25-30℃ for 36-48 h to prepare AMP-HTBC. KMnO4 (Bacillus pallida + Aspergillus mycelium balls + potassium permanganate modified walnut shell biochar + Chlorella), i.e., mycelium ball composite material. The prepared mycelium ball composite material is washed 2-3 times with ultrapure water for later use.

[0033] The functions of each component in this invention are as follows: While adding aerobic denitrifying bacteria alone can effectively remove pollutants from slightly polluted water, its effectiveness is limited by insufficient carbon sources. Therefore, modified biochar is selected as an electron shuttle (RMS) to improve the electron transfer efficiency of aerobic denitrifying bacteria and serve as a partial carbon source, alleviating the limitation of denitrification rate due to insufficient carbon sources. However, this approach suffers from the loss of the aerobic denitrifying bacteria's ecological niche and the difficulty in recovering the biochar, leading to secondary pollution. Using mycelial balls as a biological carrier provides a favorable growth environment for aerobic denitrifying bacteria and allows the biochar to be adsorbed onto the surface of the mycelial balls, eliminating the problem of secondary pollution. Finally, a certain amount of microalgae is added to provide some oxygen and carbon sources to the aerobic denitrifying bacteria through photosynthesis, thereby improving the metabolic activity and denitrification rate of the aerobic denitrifying bacteria. Furthermore, microalgae can promote the secretion of microbial extracellular polymers (EPS), thus improving the overall structural stability of the mycelial ball composite material. Moreover, the raw materials of this invention are widely available, the preparation method is feasible, and it has good engineering application value and promotion prospects.

[0034] Example 1 The preparation method of the mycelial ball composite material for enhanced denitrification of slightly polluted water according to the present invention comprises the following steps: Sampling was conducted in the landscape water body, and non-nitrate-utilizing bacteria were eliminated using nitrate-enriched culture medium, allowing denitrifying bacteria to grow. 9 ml of sample was added to 100 ml of nitrate-enriched culture medium (composition shown in Table 1), and cultured at 25℃ and 180 rpm on a shaker. After 36 h of enrichment culture, the culture was repeated twice, and the resulting bacterial solution was serially diluted (10⁻⁶ ppm). -4 -10 -7 After being spread onto a culture medium and placed in a constant temperature incubator, two different colonies grew on the culture medium plate after 36 hours: bacterial colonies and fungal colonies.

[0035] The bacterial strain was identified by testing the performance of sterile aerobic denitrification liquid culture medium and culturing at 30℃ and 170 rpm in a shaker. Ochrobactrum (Genus Paleobacterium).

[0036] Preparation of *Alanobacterium* suspension: Take 15 ml of aerobic denitrification liquid culture medium containing *Alanobacterium* cultured for 36 h, centrifuge at 5000 r / min for 10 min, discard the supernatant to obtain *Alanobacterium* suspension.

[0037] Preparation of fungal suspension: Using an inoculation loop, a suitable amount of fungal cells were picked from the culture medium plate and identified as Aspergillus. A suitable amount of fungal spores were added to the fungal mycelial ball culture medium. The composition of the fungal mycelial ball culture medium is shown in Table 3. 15 ml of the fungal mycelial ball liquid culture medium containing Aspergillus fungus, which has been cultured for 36 h, was taken and centrifuged at 6000 r / min for 5 min. The supernatant was discarded to obtain the Aspergillus fungal suspension.

[0038] Add 0.5 ml of fungal suspension to the fungal mycelial ball culture medium. The composition of the fungal mycelial ball culture medium is shown in Table 3. Incubate at 25℃ and 180 r / min on a shaker to obtain Aspergillus mycelial balls.

[0039] Add 0.5 ml (OD600=1.0) of *Bacillus cereus* suspension to the culture medium containing *Aspergillus* mycelial balls, and incubate for 36 h at 170 rpm and 25°C in a shaker to prepare DMP (*Bacillus cereus* + *Aspergillus* mycelial balls). Add 4.5 ml of modified biochar suspension to the DMP culture medium, along with 4.5 ml (OD600=1.0) of *Chlorella vulgaris* suspension, and incubate for 36 h at 170 rpm and 25°C in a shaker to prepare AMP-HTBC. KMnO4 (Bacillus pallida + Aspergillus fungal mycelium + potassium permanganate modified walnut shell biochar + Chlorella).

[0040] Example 2 The preparation method of the mycelial ball composite material for enhanced denitrification of slightly polluted water according to the present invention comprises the following steps: Sampling was conducted in the landscape water body, and non-nitrate-utilizing bacteria were eliminated using nitrate-enriched culture medium, allowing denitrifying bacteria to grow. 10 ml of sample was added to 150 ml of nitrate-enriched culture medium (components shown in Table 1 below), and cultured at 30℃ and 170 rpm on a shaker. After 48 h of enrichment culture, the culture was repeated twice, and the resulting bacterial solution was serially diluted (10⁻⁶ ppm). -4 -10 -7 After being spread onto a culture medium, the culture was placed in a constant temperature incubator. After 48 hours, two different colonies grew on the culture medium plate: bacterial colonies and fungal colonies.

[0041] The bacterial strain was identified by testing the performance of sterile aerobic denitrification liquid culture medium and culturing at 25℃ and 180 rpm on a shaker. Ochrobactrum (Genus Paleobacterium).

[0042] Preparation of *Alamydia spp.* suspension: Take 20 ml of aerobic denitrification liquid culture medium containing *Alamydia spp.* cultured for 48 h, centrifuge at 6000 r / min for 5 minutes, discard the supernatant, and obtain the *Alamydia spp.* suspension.

[0043] Preparation of fungal suspension: Using an inoculation loop, a suitable amount of fungal cells were picked from the culture medium plate and identified as Aspergillus. A suitable amount of fungal spores were added to the fungal mycelial ball culture medium. The composition of the fungal mycelial ball culture medium is shown in Table 3. 20 ml of the fungal mycelial ball liquid culture medium containing Aspergillus fungus, which has been cultured for 36 h, was taken and centrifuged at 5000 r / min for 10 min. The supernatant was discarded to obtain the Aspergillus fungal suspension.

[0044] Add 1 ml of fungal suspension to the fungal mycelial ball culture medium. The composition of the fungal mycelial ball culture medium is shown in Table 3. Incubate at 25-30℃ and 170-180 r / min on a shaker to obtain Aspergillus mycelial balls.

[0045] 0.6 ml (OD600=1.0) of *Aspergillus* suspension was added to the culture medium containing *Aspergillus* mycelial balls, and the medium was incubated in a shaker at 172 rpm and 26°C for 38 h to prepare DMP (*Aspergillus* + *Aspergillus* mycelial balls). 4.6 ml of modified biochar suspension was added to the DMP medium, along with 4.6 ml (OD600=1.0) of *Chlorella* suspension, and the medium was incubated in a shaker at 172 rpm and 26°C for 38 h to prepare AMP-HTBC. KMnO4 (Bacillus pallida + Aspergillus fungal mycelium + potassium permanganate modified walnut shell biochar + Chlorella).

[0046] Performance tests were conducted using sterilized aerobic denitrification liquid culture medium, incubated at 25°C and 180 rpm on a shaker. Example 3 The preparation method of the mycelial ball composite material for enhanced denitrification of slightly polluted water according to the present invention comprises the following steps: Sampling was conducted in the landscape water body, and non-nitrate-utilizing bacteria were eliminated using nitrate-enriched culture medium, allowing denitrifying bacteria to grow. 10 ml of sample was added to 120 ml of nitrate-enriched culture medium (components shown in Table 1 below), and cultured at 28℃ and 175 rpm on a shaker. After 42 h of enrichment culture, the culture was repeated twice, and the resulting bacterial solution was serially diluted (10⁻⁶ ppm). -4 -10 -7 After being spread onto a culture medium and placed in a constant temperature incubator, two different colonies grew on the culture medium plate after 42 hours: bacterial colonies and fungal colonies.

[0047] The bacterial strain was identified by testing the performance of sterile aerobic denitrification liquid culture medium and culturing at 28℃ and 175 r / min on a shaker. Ochrobactrum (Genus Paleobacterium).

[0048] Preparation of *Alamydia spp.* suspension: Take 18 ml of aerobic denitrification liquid culture medium containing *Alamydia spp.* cultured for 42 h, centrifuge at 5500 r / min for 8 min, discard the supernatant to obtain *Alamydia spp.* suspension.

[0049] Preparation of fungal suspension: Using an inoculation loop, a suitable amount of fungal cells were picked from the culture medium plate and identified as Aspergillus. A suitable amount of fungal spores were added to the fungal mycelial ball culture medium. The composition of the fungal mycelial ball culture medium is shown in Table 3. 17 ml of the fungal mycelial ball liquid culture medium containing Aspergillus fungus, which has been cultured for 42 h, was taken and centrifuged at 5500 r / min for 7 min. The supernatant was discarded to obtain the Aspergillus fungal suspension.

[0050] Add 0.8 ml of fungal suspension to the fungal mycelial ball culture medium. The composition of the fungal mycelial ball culture medium is shown in Table 3. Incubate at 25-30℃ and 170-180 r / min on a shaker to obtain Aspergillus mycelial balls.

[0051] 0.7 ml (OD600=1.0) of *Bacillus cereus* suspension was added to the culture medium containing *Aspergillus* mycelial balls, and the medium was incubated in a shaker at 174 rpm and 27°C for 40 h to prepare DMP (*Bacillus cereus* + *Aspergillus* mycelial balls). 4.7 ml of modified biochar suspension was added to the DMP culture medium, along with 4.7 ml (OD600=1.0) of *Chlorella vulgaris* suspension, and the medium was incubated in a shaker at 174 rpm and 27°C for 40 h to prepare AMP-HTBC. KMnO4 (Bacillus pallida + Aspergillus fungal mycelium + potassium permanganate modified walnut shell biochar + Chlorella).

[0052] Example 4 The preparation method of the mycelial ball composite material for enhanced denitrification of slightly polluted water according to the present invention comprises the following steps: Sampling was conducted in the landscape water body, and non-nitrate-utilizing bacteria were eliminated using nitrate-enriched culture medium, allowing denitrifying bacteria to grow. 10 ml of sample was added to 100 ml of nitrate-enriched culture medium (composition shown in Table 1), and cultured at 28℃ and 180 rpm on a shaker. After 42 h of enrichment culture, the culture was repeated twice, and the resulting bacterial solution was serially diluted (10⁻⁶ ppm). -4 -10 -7 After being spread onto a culture medium and placed in a constant temperature incubator, two different colonies grew on the culture medium plate after 42 hours: bacterial colonies and fungal colonies.

[0053] The bacterial strain was identified by testing the performance of sterile aerobic denitrification liquid culture medium and culturing at 30℃ and 170 rpm in a shaker. Ochrobactrum (Genus Paleobacterium).

[0054] Preparation of *Alamydia spp.* suspension: Take 15 ml of aerobic denitrification liquid culture medium containing *Alamydia spp.* cultured for 42 h, centrifuge at 5000 r / min for 10 min, discard the supernatant to obtain *Alamydia spp.* suspension.

[0055] Preparation of fungal suspension: Using an inoculation loop, a suitable amount of fungal cells were picked from the culture medium plate and identified as Aspergillus. A suitable amount of fungal spores were added to the fungal mycelial ball culture medium. The composition of the fungal mycelial ball culture medium is shown in Table 3. 15 ml of the fungal mycelial ball liquid culture medium containing Aspergillus fungus, which has been cultured for 42 h, was taken and centrifuged at 6000 r / min for 5 min. The supernatant was discarded to obtain the Aspergillus fungal suspension.

[0056] Add 0.5 ml of fungal suspension to the fungal mycelial ball culture medium. The composition of the fungal mycelial ball culture medium is shown in Table 3. Incubate at 25℃ and 180 r / min on a shaker to obtain Aspergillus mycelial balls.

[0057] 0.8 ml (OD600=1.0) of *Bacillus cereus* suspension was added to the culture medium containing *Aspergillus* mycelial balls, and the medium was incubated in a shaker at 176 rpm and 28°C for 42 h to prepare DMP (*Bacillus cereus* + *Aspergillus* mycelial balls). 4.8 ml of modified biochar suspension was added to the DMP culture medium, along with 4.8 ml (OD600=1.0) of *Chlorella vulgaris* suspension, and the medium was incubated in a shaker at 176 rpm and 28°C for 42 h to prepare AMP-HTBC. KMnO4 (Bacillus pallida + Aspergillus fungal mycelium + potassium permanganate modified walnut shell biochar + Chlorella).

[0058] Example 5 The preparation method of the mycelial ball composite material for enhanced denitrification of slightly polluted water according to the present invention comprises the following steps: Sampling was conducted in the landscape water body, and non-nitrate-utilizing bacteria were eliminated using nitrate-enriched culture medium, allowing denitrifying bacteria to grow. 10 ml of sample was added to 120 ml of nitrate-enriched culture medium (composition shown in Table 1), and cultured at 30℃ and 180 rpm on a shaker. After 48 h of enrichment culture, the culture was repeated twice, and the resulting bacterial solution was serially diluted (10⁻⁶ ppm). -4 -10 -7 After being spread onto a culture medium and placed in a constant temperature incubator, two different colonies grew on the culture medium plate after 36 hours: bacterial colonies and fungal colonies.

[0059] The bacterial strain was identified by testing the performance of sterile aerobic denitrification liquid culture medium and culturing at 30℃ and 170 rpm in a shaker. Ochrobactrum (Genus Paleobacterium).

[0060] Preparation of *Alamydia spp.* suspension: Take 18 ml of aerobic denitrification liquid culture medium containing *Alamydia spp.* cultured for 36 h, centrifuge at 5500 r / min for 8 min, discard the supernatant to obtain *Alamydia spp.* suspension.

[0061] Preparation of fungal suspension: Using an inoculation loop, a suitable amount of fungal cells were picked from the culture medium plate and identified as Aspergillus. A suitable amount of fungal spores were added to the fungal mycelial ball culture medium. The composition of the fungal mycelial ball culture medium is shown in Table 3. 15 ml of the fungal mycelial ball liquid culture medium containing Aspergillus fungus, which has been cultured for 36 h, was taken and centrifuged at 6000 r / min for 5 min. The supernatant was discarded to obtain the Aspergillus fungal suspension.

[0062] Add 0.8 ml of fungal suspension to the fungal mycelial ball culture medium. The composition of the fungal mycelial ball culture medium is shown in Table 3. Incubate at 25℃ and 180 r / min on a shaker to obtain Aspergillus mycelial balls.

[0063] 0.9 ml (OD600=1.0) of *Bacillus cereus* suspension was added to the culture medium containing *Aspergillus* mycelial balls, and the medium was incubated in a shaker at 178 rpm and 29°C for 44 h to prepare DMP (*Bacillus cereus* + *Aspergillus* mycelial balls). 4.9 ml of modified biochar suspension was added to the DMP culture medium, along with 4.9 ml (OD600=1.0) of *Chlorella vulgaris* suspension, and the medium was incubated in a shaker at 178 rpm and 29°C for 44 h to prepare AMP-HTBC. KMnO4 (Bacillus pallida + Aspergillus fungal mycelium + potassium permanganate modified walnut shell biochar + Chlorella).

[0064] Example 6 The preparation method of the mycelial ball composite material for enhanced denitrification of slightly polluted water according to the present invention comprises the following steps: Sampling was conducted in the landscape water body, and non-nitrate-utilizing bacteria were eliminated using nitrate-enriched culture medium, allowing denitrifying bacteria to grow. 10 ml of sample was added to 150 ml of nitrate-enriched culture medium (composition shown in Table 1), and cultured at 30℃ and 180 rpm on a shaker. After 48 h of enrichment culture, the culture was repeated twice, and the resulting bacterial solution was serially diluted (10⁻⁶ ppm). -4 -10 -7After being spread onto a culture medium, the culture was placed in a constant temperature incubator. After 48 hours, two different colonies grew on the culture medium plate: bacterial colonies and fungal colonies.

[0065] The bacterial strain was identified by testing the performance of sterile aerobic denitrification liquid culture medium and culturing at 30℃ and 180 rpm in a shaker. Ochrobactrum (Genus Paleobacterium).

[0066] Preparation of Aristolochic Bacillus suspension: Take 15 ml of aerobic denitrification liquid culture medium containing Aristolochic Bacillus that has been cultured for 48 h, centrifuge at 6000 r / min for 10 min, discard the supernatant to obtain Aristolochic Bacillus suspension.

[0067] Preparation of fungal suspension: Using an inoculation loop, a suitable amount of fungal cells were picked from the culture medium plate and identified as Aspergillus. A suitable amount of fungal spores were added to the fungal mycelial ball culture medium. The composition of the fungal mycelial ball culture medium is shown in Table 3. 15 ml of the fungal mycelial ball liquid culture medium containing Aspergillus fungus, which has been cultured for 36 h, was taken and centrifuged at 6000 r / min for 10 min. The supernatant was discarded to obtain the Aspergillus fungal suspension.

[0068] Add 1 ml of fungal suspension to the fungal mycelial ball culture medium. The composition of the fungal mycelial ball culture medium is shown in Table 3. Incubate at 30℃ and 180 r / min on a shaker to obtain Aspergillus mycelial balls.

[0069] Add 1.0 ml (OD600=1.0) of *Bacillus cereus* suspension to the culture medium containing *Aspergillus* mycelial balls, and incubate at 180 rpm and 30°C for 48 h in a shaker to prepare DMP (*Bacillus cereus* + *Aspergillus* mycelial balls). Add 5.0 ml of KMnO4 modified biochar suspension to the DMP culture medium, along with 5.0 ml (OD600=1.0) of *Chlorella vulgaris* suspension, and incubate at 180 rpm and 30°C for 48 h in a shaker to prepare AMP-HTBC. KMnO4 (Albacterium pallida + Aspergillus fungal mycelium balls + potassium permanganate modified walnut shell biochar + Chlorella) mycelium ball composite material.

[0070] The subsequent experiments were beaker experiments, using the mycelial ball composite material (AMP-HTBC) prepared in Example 6. KMnO4The mycelial ball composite material was added to an Erlenmeyer flask containing simulated wastewater (the composition of which is shown in Table 4). The flask was then cultured in a shaker at 170-180 r / min and 25-30℃ for 2 days, with the removal efficiency of nitrates and total nitrogen measured every 12 hours. The prepared mycelial ball composite material was then added to a sequencing batch reactor (SBR) containing actual wastewater (sampled from surface water; the influent quality is shown in Table 5). The removal efficiency of nitrates and total nitrogen was measured every 12 hours.

[0071] Table 4. Simulated Wastewater Composition Table

[0072] Table 5 Influent Water Quality Table

[0073] Figure 2 This illustrates the changes in nitrate concentration and removal rate of the mycelial ball composite material from Example 6 of this invention in simulated wastewater. Under low C / N ratio conditions of 2 in the influent, AMP-HTBC... KMnO4 The nitrate removal rate reached 87.6% after 48 hours, and the final nitrate concentration in the effluent was only 0.31 mg / L. This represents a 15% improvement over the previous study which only inoculated aerobic denitrifying bacteria and mycelial ball carriers for nitrogen removal in simulated slightly polluted water. The enhanced removal rate indicates that the mycelial ball composite material of this invention has a good removal effect on nitrate in slightly polluted water.

[0074] Figure 3 This illustrates the changes in total nitrogen concentration and removal rate of the mycelial ball composite material from Example 6 of the present invention in simulated wastewater. The influent TN concentration was 4.5 mg / L, and the AMP-HTBC... KMnO4 The total nitrogen removal rate reached 82.6% after 48 hours, and the final total nitrogen concentration in the effluent was 0.78 mg / L, which can significantly improve the nitrogen removal efficiency in slightly polluted water.

[0075] Figure 4 This diagram illustrates the changes in ETSA (Electron Transport System Activity) of the mycelial ball composite material from Example 6 of this invention in simulated wastewater. This indicator represents the metabolic activity and electron transport capacity of the microorganisms in the system. AMP-HTBC KMnO4 The initial ETSA was 9.53 mg INTF / (gh), which reached 31.58 mg INTF / (gh) by the end of the 48-hour reaction, an increase of 231%, indicating that the mycelium ball composite material of the present invention has good metabolic activity in slightly polluted water.

[0076] Figure 5 This illustrates the variation in nitrate concentration and removal rate of the mycelial ball composite material from Example 6 of this invention in actual wastewater. The influent nitrate concentration of the actual wastewater was 3.12 mg / L, and the AMP-HTBC... KMnO4 The nitrate removal rate reached 79.5% after 48 hours, and the final nitrate concentration in the effluent was only 0.64 mg / L. This represents a 9% improvement over the removal rate of biological denitrification in actual slightly polluted water in existing cases. The enhanced removal rate indicates that the mycelium ball composite material of this invention also has a good removal effect on nitrate in actual slightly polluted water.

[0077] Figure 6 This illustrates the changes in total nitrogen concentration and removal rate of the mycelial ball composite material from Example 6 of the present invention in actual wastewater. The influent TN concentration of the actual wastewater was 4.85 mg / L, and the AMP-HTBC... KMnO4 The total nitrogen removal rate reached 77.1% after 48 hours, and the final total nitrogen concentration in the effluent was 1.11 mg / L.

[0078] Figure 7 This illustrates the variation of ETSA in actual wastewater using the mycelial ball composite material of Example 6 of the present invention. AMP-HTBC KMnO4 The initial ETSA was 7.15 mg INTF / (gh), which reached 28.65 mg INTF / (gh) by the end of the 48-hour reaction. This indicates that the mycelial ball composite material of the present invention also has good metabolic activity and electron transfer capacity in actual slightly polluted water bodies, providing a more favorable physiological basis for biological denitrification.

Claims

1. A method for preparing mycelial ball composite materials for enhanced denitrification of slightly polluted water, characterized in that, The steps are as follows: Bacterial and fungal strains were screened to obtain bacterial and fungal strains, and bacterial suspensions were prepared. Culture fungi to produce mycelial balls; DMP was prepared by inoculating bacterial suspensions into mycelial ball culture medium; Modified biochar was prepared by modifying biochar with KMnO4, and a modified biochar suspension was prepared. Modified biochar suspension and Chlorella suspension were added to DMP and cultured by shaking to obtain mycelial ball composite material.

2. The method for preparing mycelial ball composite material for enhanced denitrification of slightly polluted water according to claim 1, characterized in that, The bacterial strain is *Bacillus cereus*, and the fungal strain is *Aspergillus*.

3. The method for preparing mycelial ball composite material for enhanced denitrification of slightly polluted water according to claim 1, characterized in that, The bacterial and fungal strains were obtained by sampling and screening water bodies, enriching them with nitrate culture medium, and then diluting them in a gradient and spreading process.

4. The method for preparing mycelial ball composite material for enhanced denitrification of slightly polluted water according to claim 1, characterized in that, The bacteria were confirmed through denitrification performance testing. The bacterial suspension was prepared by taking 15-20 ml of aerobic denitrification liquid culture medium containing bacterial genus and incubating for 36-48 h, centrifuging at 5000-6000 r / min for 5-10 min, and discarding the supernatant to obtain the bacterial suspension.

5. The method for preparing mycelial ball composite material for enhanced denitrification of slightly polluted water according to claim 1, characterized in that, The mycelial balls were prepared by culturing fungi in a medium containing NH4Cl, glucose, KH2PO4, and MgSO4·7H2O at 25-30℃ and 170-180 r / min.

6. The method for preparing mycelial ball composite material for enhanced denitrification of slightly polluted water according to claim 1, characterized in that, The DMP preparation involves adding 0.5-1.0 ml of bacterial suspension with OD600=1.0 to the mycelial ball culture medium and culturing at 25-30℃ and 170-180 r / min for 36-48 h.

7. The method for preparing mycelial ball composite material for enhanced denitrification of slightly polluted water according to claim 1, characterized in that, The biochar used is walnut shell biochar, which is ground through a 100-mesh sieve, treated with 2% KMnO4 solution at 90-100℃ for 20-24 hours by stirring, washed until neutral, and dried at 60-70℃ for 10-12 hours to obtain modified biochar. The modified biochar suspension is prepared by dissolving 1.5-2.0g of modified biochar in 15-20ml of ultrapure water, sonicating for 10-15min, and letting stand for 3-4h.

8. The method for preparing mycelial ball composite material for enhanced denitrification of slightly polluted water according to claim 1, characterized in that, The composite culture conditions are 25-30℃, 170-180r / min shaking culture for 36-48h, the amount of Chlorella suspension added is 4.5-5ml with OD600=1.0, and the amount of modified biochar suspension added is 4.5-5ml.

9. The method for preparing mycelial ball composite material for enhanced denitrification of slightly polluted water according to claim 1, characterized in that, After the composite material is prepared, it is washed 2-3 times with ultrapure water.

10. A mycelial ball composite material for enhanced denitrification of slightly polluted water, characterized in that, The mycelial ball composite material for enhanced denitrification of slightly polluted water, as described in any one of claims 1 to 9, is prepared by the method for preparing the mycelial ball composite material for enhanced denitrification of slightly polluted water.

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