NL2036774A

NL2036774A - Co-culture product of aerobic denitrifying fungi, preparation method and application thereof

Info

Publication number: NL2036774A
Application number: NL2036774A
Authority: NL
Inventors: Li Haiyun; Li Anyi; Ma Ben; Zhang Haihan; Pan Sixuan
Original assignee: Univ Xian Architectur & Tech
Priority date: 2023-09-12
Filing date: 2024-01-10
Publication date: 2024-02-02
Also published as: CN117417838A

Abstract

The present invention provides a co-culture product of aerobic denitrifying fungi, preparation method and application thereof, wherein the co-culture product comprises T richoderma aﬂoharzianum H1 and Aspergillus niger C1, the preparation method 5 comprises 3 steps of strain suspension preparation, seed inoculation solution preparation and the co-culture product preparation; and the co-culture product of the aerobic denitrifying fungi is able to used for in-situ remediation of reservoir water bodies. Compared with separate cultures of the two strains of fungi, nitrate nitrogen removal rate of the co-culture product of the two strains of aerobic denitrifying fungi is greatly lO improved, and when used for in-situ remediation of reservoir water bodies, nitrate removal rate and nitrogen removal rate of the co-culture product of the two strains of fungi are both greatly improved.

Description

Co-culture product of aerobic denitrifying fungi, preparation method and application thereof

Technical field

The present invention belongs to the technical field of water pollution control, relates to microbial control of water pollution, and particularly relates to a co-culture product of aerobic denitrifying fungi, preparation method and application thereof.

Background technology

Biological nitrogen removal technology has many advantages, such as high efficiency, low consumption, safety, stability and thorough nitrogen removal, and it is the most promising water body nitrogen removal technology at present, in which nitrogen removal with low carbon-nitrogen ratio is the key area of water treatment.

When the ratio of carbon to nitrogen in the source reservoir is at a low level (C/N=1/3, n= 1-3 mg/L), microorganisms do not grow well. Because the ratio of carbon to nitrogen can't be improved by adding organic carbon sources (such as methanol and acetate) to the water source reservoir, and adding carbon sources increases the cost, under this condition, aerobic denitrifying microorganisms with poor nutrition can provide a new solution for in-situ restoration of the water source reservoir.

At present, most researches on the aerobic denitrifying microorganisms focus on screening and application of single-strain aerobic denitrifying microorganisms. For example, the Chinese patent with publication number of CN116355761A gives a new method of aerobic denitrifying fungi for treating slightly polluted water with low carbon and nitrogen ratio, which provides a solution for screening aerobic denitrifying fungi. In addition, a few studies have reported co-culture of bacteria and algae. For example, the

Chinese application publication number of CN108483638A discloses a method of co- culture of microorganisms to promote stable and rapid denitrification process. In this method, G.sulfurrenducens, an electricity-producing bacterium, and denitrifying bacteria obtained after domestication with formaldehyde are co-cultured, and nitrate in the culture medium is removed by using sodium acetate and nitrate as electron donors and acceptors at the carbon-nitrogen ratio of 1-9.

The prior art mentioned above mainly has following defects:

First, although a single fungus can achieve a good nitrogen removal effect in slightly polluted water with low carbon-nitrogen ratios, it is expected to further improve the nitrogen removal effect if multiple aerobic denitrifying fungi are co-cultured. However, there is no public report about it yet.

Second, although the co-culture with bacteria as a main component can realize rapid denitrification process and it is expected to achieve better denitrification effect, compared with fungi, bacteria have poor resistance to toxic compounds and harsh natural environment because of simple cell wall components thereof, and are not suitable for the in-situ remediation of reservoir water bodies.

Summary of the invention

Aiming at the shortcomings of the prior art, the present invention aims to provide a co-culture product of aerobic denitrifying fungi, preparation method and application thereof, and solve the technical problem that water body remediation method in the prior art using aerobic denitrifying fungi under the condition of low carbon-nitrogen ratio needs to be further improved.

In order to solve the technical problem, the present invention adopts the following technical solutions:

A co-culture product of aerobic denitrifying fungi comprises two strains of the aerobic denitrifying fungi; one strain of the fungi named 7richoderma afroharzianum

HI is deposited in China Center for Type Culture Collection (CCTCC), with the deposit number of CCTCC M20231207H1,; the other strain of the fungi named Aspergillus niger

C1 1s deposited in China Center for Type Culture Collection (CCTCC), with the deposit number of CCTCC M 20231208 C1.

The present invention also has following technical characteristics: the present invention also protects a preparation method of the co-culture product of the aerobic denitrifying fungi as described above, which specifically comprises following steps: step 1: preparing a strain suspension: selecting two strains of aerobic denitrifying fungi from fungal solid culture medium, then inoculating the two aerobic denitrifying fungi into a denitrification liquid culture medium together, and culturing the two aerobic denitrifying fungi in the dark for 24-72 h at a temperature of 28-37 °C with a rotation speed of 100-200 rpm, then collecting cells by centrifugation, washing the cells with phosphate buffer solution for many times,

and finally adjusting concentration of mycelium to get the strain suspension.

Step 2: preparing seed inoculation solution: inoculating the strain suspension in step 1 into the denitrification liquid culture medium, and keeping the culture medium in the dark for 24-72 h at a temperature of 28- 37 °C with a rotation speed of 100-200 rpm to get the seed inoculation liquid.

Step 3: preparing the co-culture product; inoculating the seed inoculum in step 2 into the denitrification liquid culture medium containing a load, culturing the culture medium in the dark at a temperature of 28-37 °C with a rotation speed of 100-200 rpm, replacing the old culture medium with a fresh denitrification liquid culture medium every 1-3 days, collecting the load after 10-20 days of the culture, and washing surface of the load with water for many times, thus obtaining the co-culture product of the aerobic denitrifying fungi.

Specifically, in the step 1, the concentration of the mycelium in the strain suspension 1s 0.1-0.5 g/L.

Specifically, in the step 2, an inoculation volume ratio of the strain suspension to the denitrification liquid culture medium is (0.5-2): (7-10).

Specifically, the load is polyurethane foam.

Preferably, the temperature is 30 °C and the rotation speed is 130 rpm.

The present invention also protects the application of the aerobic denitrifying fungus co-culture product for in-situ remediation of reservoir water bodies.

Specifically, the application method comprises the following steps: inoculating the co-culture of aerobic denitrifying fungi into raw water, and continuously aerating the raw water to keep dissolved oxygen concentration in the raw water at 5-15 mg/L.

Compared with the prior art, the present invention has following technical effects: (1) Compared with separate cultures of the two strains of fungi, the co-culture of the two strains of aerobic denitrifying fungi greatly improves the nitrate nitrogen removal rate, and the two strains in the co-culture product have synergistic effect. (ii) Because fungi have stronger resistance to the environment, the aerobic denitrifying fungi co-culture product of the present invention is more suitable for the in- situ remediation of reservoir water bodies. Compared with the separate culture of two strains of fungi used for water remediation, the nitrate removal rate and nitrogen removal rate of the co-culture product of two strains of fungi are greatly improved, and the two strains in the co-culture product have synergistic effect.

(ii) The preparation method of the aerobic denitrifying fungus co-culture product of the present invention has low requirements on equipment and is easy to operate, and is suitable for most laboratories and factories. (iv) The preparation method of the co-culture product of the aerobic denitrifying fungi of the present invention adopts polyurethane foam cubes as the load of bacteria, and because the polyurethane foam has a macroporous reticular structure and a high specific surface area, it can effectively immobilize microorganisms, so that the in-situ remediation of reservoir water bodies can be carried out stably and for a long time.

Description of drawings

Fig. 1 shows phylogenetic trees of two strains of fungi. In fig. 1: (a) is the phylogenetic tree of a strain C1 and (b) is the phylogenetic tree of a strain HI.

Fig. 2 is a statistical diagram of nitrate nitrogen removal ability of the two strains of fungi. in fig. 2: (a) shows change curves of nitrate nitrogen, nitrous nitrogen, ammonia nitrogen and total nitrogen in the culture medium when strain C1 is cultured alone; (b) shows change curves of nitrate nitrogen, nitrous nitrogen, ammonia nitrogen and total nitrogen in the culture medium when strain HI is cultured alone; (c) shows change curves of nitrate nitrogen, nitrous nitrogen, ammonia nitrogen and total nitrogen in the culture medium when strains C1 and H1 are co-cultured; and (d) shows change curves of dissolved organic carbon in the culture medium when strain C1 is cultured alone, strain H1 1s cultured alone, and strains C1 and H1 are co-cultured.

Fig. 3 is a statistical diagram of electron transfer chain activity results. Fig. 3: (a) shows histograms of ATP concentration in fungi cells when strain C1 is cultured alone, strain HI is cultured alone, and strains Cl and HI are cultured together; (b) shows histograms of intracellular electron transfer activity in fungi cells when strain C1 is cultured alone, strain H1 is cultured alone, and strains C1 and HI are cultured together.

Fig. 4 is a statistical diagram of results of using two strains of fungi to control water pollution. Figure 4: (a) shows change curves of nitrate nitrogen, nitrous nitrogen, ammonia nitrogen and total nitrogen in raw water of the reservoir when strain Cl is cultured alone; (b) shows change curves of nitrate nitrogen, nitrous nitrogen, ammonia nitrogen and total nitrogen in raw water of the reservoir when strain HI is cultured alone; (c) shows change curves of nitrate nitrogen, nitrous nitrogen, ammonia nitrogen and total nitrogen in raw water of the reservoir when strains C1 and H1 are co-cultured; (d) shows change curves of the dissolved organic carbon in raw water of the reservoir when strain

C1 1s cultured alone, strain H1 is cultured alone, and strains C1 and HI are co-cultured.

The specific contents of the present invention will be further explained in detail with embodiments.

Embodiments

It should be noted that all reagents and culture media used in the present invention, unless otherwise specified, adopt reagents and culture media known in the field, such as:

Fungal solid culture medium adopts conventional fungal solid culture medium known in the prior art, and formula and preparation method thereof are as follows: peptone is 5.0 g/L, glucose is 10.0 g/L, potassium dihydrogen phosphate is 1.0 g/L, dicloran is 0.002 g/L, magnesium sulfate is 0.5 g/L, rose bengal is 0.025 g/L, chloramphenicol is 0.1 g/L and agar is 15.0 g/L; adding the above components into ultrapure water to a constant volume of 1L, stirring until the mixture is completely dissolved, then adjusting pH value to 5.6 + 0.2 (25 °C), and sterilizing at 121 °C for 30 minutes for later use. the denitrification liquid medium adopted conventional denitrification liquid medium known in the prior art, and formula and preparation method thereof are as follows: KNO: is 0.108 g/L, KH;PO: is 1.5 g/L, glucose is 0.413 g/L, MgSO, 7H,0 is 0.1 g/L, Na2HPO: 12H20 is 5.0 g/L, and trace element mother liquor is 2 mL; adding the above components into ultrapure water to a constant volume to 1L, stirring until the mixture are completely dissolved, then adjusting the pH value to 7.0-7.2, and sterilizing at 121 °C for 30 minutes for later use.

The formula and preparation method of the trace element mother liquor are as follows: 4.4 mg of ZnSO, 100 mg of ethylenediaminetetraacetic acid, 10.2 mg of

MnCl; 4H:0, 11 mg of CaCl:, 10 mg of FeSO; 7H20, 3.2 mg of CuSO4-5H:0, 2.2 mg of (NH4)sMo7024-4H>0 and 3.2 mg of CoClz-6H20; adding the above components into ultrapure water to a constant volume to 1L, stirring until the mixture is completely dissolved, then adjusting the pH value to 7.0-7.2, and sterilizing at 121 °C for 30 minutes for later use.

Specific embodiments of the present invention are given below, and it should be noted that the present invention is not limited to the following specific embodiments, and all equivalent transformations made on the basis of the technical solutions of the present application fall within the protective scope of the present invention.

Embodiment 1

The present embodiment provides a co-culture product of aerobic denitrifying fungi, comprising two strains of aerobic denitrifying fungi. One strain is named 7richoderma afroharzianum HI, which is deposited in China Center for Type Culture Collection (CCTCC) in Wuhan, China, with the deposit number CCTCC M 20231207 HI.

ITS sequence of Trichoderma afroharzianum HI is as follows: 5°-

CCTGCGGAAGGATCATTACCGAGTGCGGGTCCTTTGGGCCCAACCTCCCAT

CCGTGTCTATTGTACCCTGTTGCTTCGGCGGGCCCGCCGCTTGTCGGCCGCC

GGGGGGGCGCCTCTGCCCCCCGGGCCCGTGCCCGCCGGAGACCCCAACAC

GAACACTGTCTGAAAGCGTGCAGTCTGAGTTGATTGAATGCAATCAGTTAA

AACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGA

AATGCGATAACTAATGTGAATTGCAGAATTCAGTGAATCATCGAGTCTTTG

AACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTC

ATTGCTGCCCTCAAGCCCGGCTTGTGTGTTGGGTCGCCGTCCCCCTCTCCGG

GGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGATCCTCGAGCGT

ATGGGGCTTTGTCACATGCTCTGTAGGATTGGCCGGCGCCTGCCGACGTTT

TCCAACCATTCTTTCCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGA

ACTTAAGCATATCAAT-3’.

The other strain is named Aspergillus niger C1, which is deposited in China Center for Type Culture Collection (CCTCC) in Wuhan, China, with the deposit number

CCTCC M 20231208 C1.

ITS sequence of Aspergillus niger Cl is as follows: 5°-

ATGATATGCTTAAGTTCAGCGGGTATTCCTACCTGATCCGAGGTCAACATT

TCAGAAGTTGGGTGTTTAACGGCTGTGGACGCGCCGCGCTCCCGATGCGAG

TGTGCAAACTACTGCGCAGGAGAGGCTGCGGCGAGACCGCCACTGTATTTC

GGAGACGGCCACCGCCAAGGCAGGGCCGATCCCCAACGCCGACCCCCCGG

AGGGGTTCGAGGGTTGAAATGACGCTCGGACAGGCATGCCCGCCAGAATA

CTGGCGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCA

ATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCAGAACCAA

GAGATCCGTTGTTGAAAGTTTTGATTCATTTTCGAAACGCCTACGAGAGGC

GCCGAGAAGGCTCAGATTATAAAAAAAACCCGCGAGGGGGTATACAATAA

GAGTTTTGGTTGGTCCTCCGGCGGGCGCCTTGGTCCGGGGCTGCGACGCAC

CCGGGGCAGAGATCCCGCCGAGGCAACAGTTTGGTAACGTTCACATTGGG

TTTGGGAGTTGTAAACTCGGTAATGATCCCTCCGCAG-3’.

In the present embodiment, a screening method of the above two strains of aerobic denitrifying fungi specifically comprise following steps:

Step 1, enrichment culture:

Treating mud-water mixture collected from the water source reservoir by ultrasound with the ultrasonic power of 40 %(200 W) and the ultrasonic time of 10 s to prepare a suspension; then taking 5mL of the suspension and diluting the suspension taken to 10 times, 100 times and 1000 times by gradient dilution method; coating 100 pL of the diluted suspension on the fungal solid culture medium, and coating three plates as parallel for each dilution gradient, and placing the coated plates in a biochemical incubator at 30 °C for 5-7 days until colonies are formed.

Step 2, separation and purification: taking the plates with the colonies out of the biochemical incubator; picking out blue-green fluffy bacteria in a sterile environment, and then separating the bacteria on a new fungal solid medium using streak plate method; ensuring that the incubator is full of oxygen and making upside down and putting the plates in a 30 °C incubator for 3-5 days; repeating the above steps until the colonies on the solid plates appear clear turquoise, and appearances of the colonies are the same size, and there are no other miscellaneous bacteria; and selecting all the turquoise colonies for culture to obtain candidate fungi. In the present embodiments, 132 candidate fungi were screened.

Step 3, screening denitrification performance: the 132 candidate fungi are inoculated into a denitrification liquid culture medium for denitrification screening. In the present embodiment, two strains of fungi, named C1 and H1, were finally screened.

Step 4, strain identification: step 4.1, biological morphological identification: biological morphology of the colony of H1 showed as filamentous fungi because of a large diameter, light yellow color, and white and filamentous edge. The biological morphology of the colony of C1 showed as filamentous fungi because of a slightly smaller diameter than that of H1, dark green color, and light yellow edge.

Step 4.2, obligate aerobe identification: inoculating HI and C1 into the denitrification liquid culture medium for culture,

transferring H1 and C1 to an anaerobic bottle and filling H1 and C1 with nitrogen gas for sealing after reaching a stable period; after being cultured in an incubator at 30 °C for 7 days, if the strains show no obvious signs of growth, it means that the strains are strict aerobic denitrifying fungi. The results showed that H1 and C1 are both aerobic denitrifying fungi.

Step 4.3, molecular biological identification: performing PCR (Polymerase Chain Reaction ) with primers ITS1 and ITS4, wherein sequence of the primer ITS1 is TCCGTAGGTGAACCTGCGG, and the sequence of the primer ITS4 is TCCTCCGCTTATTGATATGC. The PCR system comprises 0.5uL Template (genomic DNA, 20-50 ng/uL), 2.5uL 10xBuffer (containing

Mg?*), 1 uL dNTP (2.5 mM each), 0.5 uL polymerase, primers ITS1 and ITS4 (10 uM) 0.5 uL each and 9.5 pL distilled water. Amplification process of PCR comprises the following: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 45s, annealing at 55 °C for 45 s, extension at 72 °C for 1 min; and repeating the above cycle 30 times, repair extension at 72 °C for 10 min, and ending the process at 4 °C.

After PCR is ended, ITS (Internal Transcribed Spacer) fragments of the fung are obtained; after sequencing, the ITS fragments of the fungi are compared with the known

ITS sequences of filamentous fungi in NCBI (National Center for Biotechnology

Information), and phylogenetic trees are established, as shown in Fig. 1. As can be seen from Figure 1, the strain HI has 88% similarity with 7richoderma afroharzianum, and the strain C1 has 87% similarity with Aspergillus niger.

In the present embodiment, functions of the two aerobic denitrifying fungi were tested as follows: (a) Nitrate nitrogen and dissolved organic carbon removal capacity test: inoculating about 2mL of a strain suspension into 200 mL conical flask containing 150 mL of the denitrification liquid culture medium; and then, inoculating the culture in a shaking incubator at 130 r/min and 30 °C for 5 days. In the experiment, the dissolved oxygen concentration should be kept close to 7 mg/L; samples are collected every 12 hours and filtered by 0.22pm filter membrane; and selecting the supernatant for analysis of concentrations of nitrate nitrogen (NO3™-N), nitrous nitrogen (NO;-N), ammonia nitrogen (NH:"-N), total nitrogen (TN) and dissolved organic carbon (DOC). The results were shown in Fig. 2.

It can be seen from Fig. 2 that contents of nitrate nitrogen and total nitrogen in the denitrification liquid medium did not change much when the strain C1 or Hl was cultured alone, but decreased greatly when the strain C1 and H1 were co-cultured. After calculation according to the data in Fig. 2, the nitrate removal rates of the strain C1 cultured alone, the strain H1 cultured alone and the strains C1 and HI1 co-cultured in logarithmic phase were 19%, 11% and 70% respectively. The above results showed that compared with when C1 or H1 was cultured alone, the nitrate removal ability was greatly improved when the strains C1 and HI were co-cultured, and there was a synergistic effect between the two strains.

In addition, according to the calculation of the data in Fig. 2, it can be known that the removal rates of the dissolved organic carbon of the strain C1 cultured alone, the strain H1 cultured alone and the strains C1 and H1 co-cultured were 72.44%, 70.22%, and 92.67% respectively. The above results showed that compared with when C1 or H1 was cultured alone, the removal rates of the dissolved organic carbon was greatly improved when the strains C1 and H1 were co-cultured, and there was a synergistic effect between the two strains. (b) Detection of electron transfer chain activity: testing activities of nitrite reductase and nitrous oxide reductase by NIR (nitrite reductase) Stain Kit and NOS (nitrous oxide) Stain Kit (producted by SolarBio, USA), and then testing the enzyme activities of fungal cells by full-wavelength multifunctional enzyme label instrument (producted by Thermo Scientific, USA), and the results were shown in Fig. 3. After calculating the data in Fig. 3, it can be seen that intracellular ATP concentration of fungi increased by nearly 1.4 times and intracellular electron transfer activity of fungi increased by 3 times after the co-culture of the strains C1 and HI, compared with when C1 or H1 was cultured alone.

Embodiment 2

The embodiment provides a preparation method of aerobic denitrifying fungi co- culture product, which specifically comprises the following steps: step 1: preparing a strain suspension: storing the two strains of aerobic denitrifying fungi in embodiment 1 on a fungal solid medium; selecting two strains of the aerobic denitrifying fungi from the fungal solid culture medium, then inoculating the two aerobic denitrifying fungi into a denitrification liquid culture medium together, and culturing the two aerobic denitrifying fungi in the dark for 48h at a temperature of 30 °C with a rotation speed of 130 rpm, then collecting cells by centrifugation, washing the cells with phosphate buffer solution for many times, and finally adjusting concentration of mycelium to 0.3g/L (dry weight) to get the strain suspension.

Step 2: preparing seed inoculation solution: inoculating 10 ml of the strain suspension in step 1 into 90 ml of a denitrification liquid culture medium, and keeping the culture medium in the dark for 48 h at a temperature of 30 °C with a rotation speed of 130 rpm to get the seed inoculation liquid.

Step 3: preparing the co-culture product; inoculating 15 ml of the seed inoculum in step 2 into a 250 mL conical flask containing 135 mL of a denitrification liquid culture medium; in the denitrification liquid culture medium, several sterilized polyurethane foam cubes are soaked; culturing the conical flask in the dark at a temperature of 30 °C with a rotation speed of 130 rpm, replacing the old culture medium in the conical flask with a fresh denitrification liquid culture medium every 2 days, collecting 5 polyurethane foam cubes after 14 days of the culture, and washing surfaces of the polyurethane foam cubes with ultrapure water for three times; bacteria attached to the polyurethane foam cubes are the aerobic denitrifying fungi co-culture product.

Embodiment 3

The present embodiment provides an application of the co-culture product of the aerobic denitrifying fungi in Embodiment 1 for in-situ remediation of reservoir water, and the application method comprises: inoculating polyurethane foam cubes with aerobic denitrifying fungi co-culture product into a 2 L beaker containing 1.5 L of regulated raw water; keeping dissolved oxygen concentration in the raw water at 9.5mg/L by using an air pump to continuously fill the beaker with oxygen to create an aerobic environment; collecting 10 mL of the raw water every day to determine concentrations of nitrate nitrogen (NO:'-N), nitrous nitrogen (NO:-N), ammonia nitrogen (NH:’-N), total nitrogen (TN) and dissolved organic carbon (DOC); and all the experiments should be repeated three times. The experimental results were shown in Fig. 4.

In the present embodiment, the raw water comes from a middle layer (30 m) of a diversion tower of Lijiahe Reservoir in December, 2022, and the sampling comprises a surface layer, middle layer and bottom layer of the water body. Water quality of the collected water sample was stabilized and transported to the laboratory within 3 hours.

S11 -

The water quality information of the raw water was shown in Table 1.

Table 1. Water quality parameters of middle layer of diversion tower of Lijiahe

Reservoir “Parameters pH Total Total Nitrate ammonia nitrous DOC Fe Mn nitrogen phosphorus nitrogen nitrogen nitrogen

Diversion 7.85 1.542 0.023 1.363 0.116 0.063 3.540 0.045 0.075 tower

Comparative example 1:

The present comparative example shows application of the single culture of the aerobic denitrifying fungus 7richoderma afroharziaman H1 in Embodiment 1 for in-situ remediation of the reservoir water. The application method is basically the same as that in Embodiment 3, with a difference that the polyurethane foam cubes attached with the single culture product of aerobic denitrifying fungus Trichoderma afroharzianum HI were inoculated into the 2 L beaker containing 1.5 L of the regulated raw water.

In the present comparative example, preparation method of the single culture of the aerobic denitrifying fungus 7richoderma afroharzianum HI is basically the same as that in Embodiment 2, with a difference that in step 1, only the aerobic denitrifying fungus

Trichoderma afroharzianum HI is selected from the fungal solid medium and then inoculated into the denitrifying liquid medium.

Comparative example 2:

The present comparative example shows application of the single culture of the aerobic denitrifying fungus Aspergillus niger C1 in Embodiment 1 for in-situ remediation of the reservoir water. The application method is basically the same as that in Embodiment 3, with a difference that the polyurethane foam cubes attached with the single culture product of aerobic denitrifying fungus Aspergillus niger Cl were inoculated into the 2 L beaker containing 1.5 L of the regulated raw water.

In the present comparative example, preparation method of the single culture of the aerobic denitrifying fungus Aspergillus niger C1 is basically the same as that in

Embodiment 2, with a difference that in step 1, only the aerobic denitrifying fungus

Aspergillus niger C1 is selected from the fungal solid medium and then inoculated into the denitrifying liquid medium.

In the present comparative example, final water pollution control results were shown in Fig. 4.

The following conclusions can be drawn from Embodiment 3, Comparative example 1 and Comparative example 2:

After calculation according to the data in Fig. 2, it can be known that nitrate residues of the strain C1 cultured alone, the strain H1 cultured alone and the strains C1 and H1 co-cultured are 1.10 mg/L, 1.25mg/L and 0.4 mg/L respectively after 7 days; the nitrate removal rates of the strain C1 cultured alone, the strain H1 cultured alone and the strains

C1 and HI co-cultured are 20.41%, 7.72% and 70.29% respectively; the total nitrogen removal rates of the strain C1 cultured alone, the strain H1 cultured alone and the strains

Cl and HI co-cultured are 24.05%, 12.66% and 73.42% respectively. The results showed that the nitrate removal ability of reservoir raw water was greatly improved after the co-culture of the strains C1 and HI, and there was a synergistic effect between the two strains.

In addition, nitrite accumulated when the strain C1 or strain H1 was cultured alone, but the accumulation was almost not observed in the co-culture system of the strain C1 and H1, which further shows that the co-culture of strain C1 and H1 can greatly improve the nitrogen removal ability of fungi in water. The present study provides a new way to improve the possibility of treating nitrate in slightly polluted water in reservoirs, and enhancement of nitrogen removal ability by the co-culture of fungi is of guiding significance for drinking water treatment.

Xi'an University of Architecture and Technology CN 2023111699080 2023-09-12 Jf

ZERBA A Xi'an University of Architecture and Technology Ki

ZHANG(surname), Haihan (given name) (male) Co-culture product of aerobic denitrifying fungi, preparation method and application thereof 4 577 DNA PAT source 1..577 mol_type other DNA organism Trichoderma afroharzianum cctgcggaaggatcattaccgagtgcgggtcctttgggcccaacctcccatccgtgtctattgtaccctgttgcttcggcg ggcccgccgcttgteggcegccgggggggcgcctctgccccccgggcccgtgcccgccggagaccccaacacgaac actgtctgaaagcgtgcagtctgagttgattgaatgcaatcagttaaaactttcaacaatggatctcttggttccggcat cgatgaagaacgcagcgaaatgcgataactaatgtgaattgcagaattcagtgaatcatcgagtctttgaacgcacat tgcgccccctggtattccggggggcatgcctgtccgagcgtcattgctgccctcaagcccggcttgtgtgttgggtcgcc gtccccctctccggggggacgggcccgaaaggcagcggcggcaccgcgtccgatcctcgagcgtatggggctttgtc acatgctctgtaggattggccggcgcctgccgacgttttccaaccattctttccaggttgacctcggatcaggtagggat acccgctgaacttaagcatatcaat 595 DNA PAT source 1..595 mol_type other DNA organism Aspergillus niger atgatatgcttaagttcagcgggtattcctacctgatccgaggtcaacatttcagaagttgggtgtttaacggctgtgga cgcgccgcgctcccgatgcgagtgtgcaaactactgcgcaggagaggctgcggcgagaccgccactgtatttcggag acggccaccgccaaggcagggccgatccccaacgccgaccccccggaggggttcgagggttgaaatgacgctcgg acaggcatgcccgccagaatactggcgggcgcaatgtgcgttcaaagattcgatgattcactgaattctgcaattcac attacttatcgcatttcgctgcgttcttcatcgatgccagaaccaagagatccgttgttgaaagttttgattcattttcgaa acgcctacgagaggcgccgagaaggctcagattataaaaaaaacccgcgagggggtatacaataagagttttggtt ggtcctccggcgggcgccttggtccggggctgcgacgcacccggggcagagatcccgccgaggcaacagtttggta acgttcacattgggtttgggagttgtaaactcggtaatgatccctccgcag 19 DNA PAT source 1..19 mol_type other DNA organism synthetic construct tccgtaggtgaacctgcgg 20 DNA PAT source 1..20 mol_type other DNA organism synthetic construct tcctccgcttattgatatgc

Claims

ConclusiesConclusions

1. Samenkweekproduct van een aerobe denitrificerende schimmel, dat twee stammen van de aerobe denitrificerende schimmels omvat; waarbij één stam van de schimmels met de naam Trichoderma afroharzianum H1 gedeponeerd is in het China Center for Type Culture Collection (CCTCC), met het depotnummer CCTCC M20231207H1; en de andere stam van de schimmel met de naam Aspergillus niger C1 gedeponeerd 1s in het China Center for Type Culture Collection (CCTCC), met het depotnummer CCTCC M 20231208 C1.1. Co-cultivation product of an aerobic denitrifying fungus, comprising two strains of the aerobic denitrifying fungi; wherein one strain of the fungi named Trichoderma afroharzianum H1 has been deposited in the China Center for Type Culture Collection (CCTCC), with the deposit number CCTCC M20231207H1; and the other strain of the fungus named Aspergillus niger C1 deposited 1s in the China Center for Type Culture Collection (CCTCC), with the deposit number CCTCC M 20231208 C1.

2. Bereidingswerkwijze van het samenkweekproduct van de aerobe denitrificerende schimmels volgens conclusie 1, die specifiek de volgende stappen omvat: stap 1: het bereiden van een stamsuspensie: het selecteren van twee stammen van aerobe denitrificerende schimmels uit vast schimmelkweekmedium, vervolgens het samen inoculeren van de twee aerobe denitrificerende schimmels in een vloeibaar denitrificatiekweekmedium en het 24-72 uur kweken van de twee aerobe denitrificerende schimmels in het donker bij een temperatuur van 28-37 ° C met een rotatiesnelheid van 100-200 rpm, en vervolgens het verzamelen van cellen door centrifugatie, het vele keren wassen van de cellen met een fosfaatbufferoplossing en ten slotte het aanpassen van de myceliumconcentratie om de stamsuspensie te verkrijgen; stap 2: bereiding van kieminoculatieoplossing: het inoculeren van de stamsuspensie uit stap 1 in het vloeibare denitrificatiekweekmedium en het 24-72 uur in het donker houden van het kweekmedium bij een temperatuur van 28-37 ° C met een rotatiesnelheid van 100-200 rpm om kieminocatievloeistof te krijgen; en stap 3: het bereiden van het samenkweekproduct; het inoculeren van het kieminoculum uit stap 2 in het vloeibare denitrificatiekweekmedium dat een lading bevat, het kweken van het kweekmedium in het donker bij een temperatuur van 28-37°C met een rotatiesnelheid van 100-200 rpm, waarbij het oude kweekmedium elke 1-3 dagen met vers vloeibaar denitrificatiekweekmedium vervangen wordt, het verzamelen van de lading na 10-20 dagen van de kweek en het vele malen wassen van het oppervlak van de lading met water, waardoor het samenkweekproduct van de aerobe denitrificerende schimmels verkregen wordt.Preparation method of the co-cultivation product of the aerobic denitrifying fungi according to claim 1, which specifically comprises the following steps: step 1: preparing a strain suspension: selecting two strains of aerobic denitrifying fungi from solid fungal culture medium, then co-inoculating the two aerobic denitrifying fungi in a liquid denitrifying culture medium and culturing the two aerobic denitrifying fungi in the dark at a temperature of 28-37 °C with a rotation speed of 100-200 rpm for 24-72 hours, and then collecting cells by centrifugation , washing the cells many times with a phosphate buffer solution and finally adjusting the mycelial concentration to obtain the stock suspension; step 2: preparation of germination inoculation solution: inoculating the stock suspension from step 1 into the liquid denitrification culture medium and keeping the culture medium in the dark for 24-72 hours at a temperature of 28-37 °C with a rotation speed of 100-200 rpm to to obtain germination inoculation fluid; and step 3: preparing the co-cultivation product; inoculating the germination inoculum from step 2 into the liquid denitrification culture medium containing a charge, culturing the culture medium in the dark at a temperature of 28-37°C with a rotation speed of 100-200 rpm, changing the old culture medium every 1- 3 days with fresh liquid denitrification culture medium, collecting the batch after 10-20 days of culture and washing the surface of the batch with water many times, thereby obtaining the co-cultivation product of the aerobic denitrifying fungi.

3. Bereidingswerkwijze van het samenkweekproduct van de aerobe denitrificerende schimmels volgens conclusie 2, waarbij in stap 1 de concentratie van het mycelium in de stamsuspensie 0,1-0,5 g/L is.Preparation method of the co-cultivation product of the aerobic denitrifying fungi according to claim 2, wherein in step 1 the concentration of the mycelium in the stock suspension is 0.1-0.5 g/L.

4. Bereidingswerkwijze van het samenkweekproduct van de aerobe denitrificerende schimmels volgens conclusie 2, waarbij in stap 2 een inoculatievolumeverhouding van de stamsuspensie tot het vloeibare denitrificatiekweekmedium (0,5-2):(7-10) is.The preparation method of the co-cultivation product of the aerobic denitrifying fungi according to claim 2, wherein in step 2 an inoculation volume ratio of the stock suspension to the liquid denitrification culture medium is (0.5-2):(7-10).

5. Bereidingswerkwijze van het samenkweekproduct van de aerobe denitrificerende schimmels volgens conclusie 2, waarbij de lading polyurethaanschuim is. 6. 3.Preparation method of the co-cultivation product of the aerobic denitrifying fungi according to claim 2, wherein the charge is polyurethane foam. 6. 3.

6. Bereidingswerkwijze van het samenkweekproduct van de aerobe denitrificerende schimmels volgens conclusie 2, waarbij de temperatuur 30°C is en de rotatiesnelheid 130 rpm is.Preparation method of the co-cultivation product of the aerobic denitrifying fungi according to claim 2, wherein the temperature is 30°C and the rotation speed is 130 rpm.

7. Samenkweekproduct van de aerobe denitrificerende schimmel volgens conclusie 1 is van toepassing op ix sifu-sanering van waterreservoirlichamen.The co-cultivation product of the aerobic denitrifying fungus according to claim 1 is applicable to ix siphon remediation of water reservoir bodies.

8. Toepassing volgens conclusie 7, waarbij een werkwijze daarvan het volgende omvat: het inoculeren van de aerobe denitrificerende schimmels in onbehandeld water, en het ononderbroken beluchten van het onbehandelde water om opgeloste zuurstofconcentratie in het onbehandelde water op 5-15 mg/L te houden.Use according to claim 7, wherein a method thereof comprises: inoculating the aerobic denitrifying fungi into raw water, and continuously aerating the raw water to maintain dissolved oxygen concentration in the raw water at 5-15 mg/L .