Polyacrylamide degrading fungus as well as screening and domestication method and application thereof

Abstract

The invention belongs to the technical field of environmental protection, and relates to screening, domestication and application of focused polyacrylamide (PAM) for efficiently degrading fungi. The strain (# 12) is preserved in CGMCC (China General Microbiological Culture Collection Center) and is trichoderma asperellum, and the similarity between the ITS sequence of the strain and the gene reaches 99%. Aiming at soil hardening and acrylamide pollution caused by difficult degradation of non-ionic PAM in bauxite slime, the inventor adopts a gradient concentration screening and multi-stage domestication process. The method comprises the following steps: carrying out enrichment culture on slurry containing PAM, carrying out gradient screening in a basic solution with a specific concentration, separating and purifying to obtain an optimal strain, and carrying out continuous passage domestication through a six-stage culture medium, so that the degradation rate is increased by 23.45%. Under optimized conditions, the PAM degradation rate of the strain reaches 64.18%, the viscosity is reduced by 88.03%, degradation is achieved through physical destruction and enzymatic hydrolysis, and no acrylamide monomer is detected. The strain has activity at the temperature of 25-45 DEG C and the pH value of 4-8, is suitable for bioremediation of PAM-containing slime, alleviation of soil hardening and elimination of risks of toxic and side products, reveals the efficient degradation characteristic of trichoderma fungi to nonionic PAM for the first time, and assists in recycling of mining wastes.

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental protection, and particularly relates to a polyacrylamide-degrading fungus, a method for screening and domestication thereof, and an application thereof. Background Art

[0002] Polyacrylamide (PAM) has high water solubility, high viscosity, and effective flocculation effect. These characteristics make PAM widely used in the mining field. After adding PAM to washing mine wastewater for flocculation, sedimentation, and compression, the moisture content and volume of mine sludge can be greatly reduced. The PAM-containing mine sludge can be used for reclamation after modification treatment, which can effectively solve the problem of insufficient tailing soil sources and realize the safe disposal of mine sludge and the utilization of land resources. However, since PAM often forms a massive body with the concentrated mine sludge, this structural characteristic will cause soil compaction and make it difficult for plants to take root. At the same time, PAM will produce toxic acrylamide under natural conditions, which will not only pollute rivers and soils in the environment but also cause serious harm to human health. It is reported that the degradation methods of PAM include physical treatment, chemical oxidation, and microbial degradation, among which microbial degradation has the characteristics of low cost and no secondary pollution. Summary of the Invention

[0003] The purpose of the present invention is to provide a polyacrylamide-degrading fungus, a method for screening and domestication thereof, and an application thereof, which can significantly improve the degradation rate of PAM by the degrading fungus, and after optimizing the degradation conditions, can significantly reduce the concentration and molecular weight of polyacrylamide in mine sludge, reduce the viscosity of PAM, thereby reducing the impact of mine sludge compaction on plant rooting and reducing the harm of acrylamide to human life safety and health.

[0004] The technical solution of the present invention is as follows:

[0005] A highly efficient polyacrylamide-degrading bacterium, which is preserved in the China General Microbiological Culture Collection Center (CGMCC), with the preservation number: CGMCC No. 27890, the preservation date: July 14, 2023, the preservation address: Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, Postcode: 100101, and the taxonomic name is Alcaligenes faecalis, and the strain number is EPDB-5.

[0006] For the polyacrylamide-degrading fungus with the strain number #12, through BLAST alignment of the ITS sequence of this strain, it is found that #12 has at least 99% similarity with the gene sequence of Trichoderma asperellum-like.

[0007] The polyacrylamide-degrading fungus is screened from bauxite mine sludge; the polyacrylamide is non-ionic polyacrylamide.

[0008] The present invention also provides a method for screening and domestication of the polyacrylamide-degrading fungus, and the specific steps are as follows:

[0009] (1) Collect bauxite ore sludge containing polyacrylamide as the inoculation source, add the bauxite ore sludge containing polyacrylamide to the enrichment medium, and perform constant-temperature shaking culture for 4 days;

[0010] (2) Inoculate the bacterial suspension obtained in step (1) into the basic liquid medium, so that the polyacrylamide concentration in the medium is successively 100 ppm to 500 ppm;

[0011] (3) Dilute the bacterial suspension obtained in step (2) in proportion and evenly coat it on the Martin's medium, and perform constant-temperature culture to obtain multiple strains of fungi with different morphologies;

[0012] (4) Inoculate the multiple strains of fungi obtained in step (3) into the Martin's medium by the method of continuous streaking, and perform constant-temperature purification culture;

[0013] (5) Prepare spore suspensions from the purified fungi obtained in step (4), inoculate them into the polyacrylamide medium, culture for 8 days, and then measure their respective polyacrylamide degradation abilities to obtain the optimal degrading fungus.

[0014] (6) Add the spore liquid of the optimal PAM-degrading fungus screened in step (5) to the No. ① medium of the domestication medium, perform constant-temperature shaking culture for 4 days, then pour out half of the supernatant, and continue to add the same volume of the No. ① medium, and continue to culture under the same conditions for 4 days.

[0015] (7) Completely pour out the supernatant after the culture in step (6), add the bacterial cells to the No. ② medium, and culture according to the methods of step (6) and step (7) until it is transferred to the No. ⑥ medium.

[0016] (8) Inoculate the fungus in the No. ⑥ medium in step (7) onto the Martin's medium, and after it grows to maturity, obtain the spore suspension of the optimally domesticated fungus according to the method of step (5).

[0017] Add the spore liquids of the optimally domesticated polyacrylamide-degrading fungus before and after domestication to the basic liquid medium, perform constant-temperature shaking culture for 8 days. After domestication, the degradation rate of the degrading fungus to PAM increases by 23.45%.

[0018] Add the spore liquid of the optimally domesticated polyacrylamide-degrading fungus to the basic liquid medium, the inoculation amount is between 1% and 6%, the optimal inoculation amount is 5%, the pH is between 4 - 8, the optimal pH is 5, the temperature is between 25°C and 40°C, and the optimal temperature is 30°C.

[0019] The advantages and technical effects of the present invention are as follows:

[0020] The present invention has successfully screened out a strain of polyacrylamide-degrading fungus. After domestication, the degradation rate of PAM is increased by 23.45%. This domestication method can greatly improve the degradation ability of the fungus to PAM. It is found that the hyphae produced by the polyacrylamide-degrading fungus during the degradation process can physically damage the surface structure of polyacrylamide, and then degrade polyacrylamide through enzymatic reactions. After optimizing the degradation conditions, it is found that the viscosity reduction rate of this fungus to PAM reaches 88.03%, which is much higher than the previous research on polyacrylamide-degrading bacteria. Such a phenomenon has not been reported in previous polyacrylamide-degrading bacteria, and it can solve problems such as the high viscosity of PAM, the long natural degradation time, and the generation of acrylamide by abiotic degradation.

[0021] The degrading fungus screened out in the present invention is derived from bauxite ore sludge and obtained after systematic domestication. Brief Description of the Drawings

[0022] Figure 1 It is a morphological characteristic diagram of the fungus of the present invention.

[0023] Figure 2 It is a phylogenetic tree diagram of the fungus of the present invention.

[0024] Figure 3 It is a diagram of the degradation rate of PAM by the fungus before and after domestication.

[0025] Figure 4 Optimization diagram of the fungus degradation conditions; among them, (a) is the biodegradation rate of PAM according to the change of pH value; (b) is the biodegradation rate of PAM according to the change of inoculum size; (c) is the biodegradation rate of PAM according to the change of temperature.

[0026] Figure 5 It is a scanning electron microscope diagram of PAM before and after degradation; among them, (left) is the scanning electron microscope diagram of PAM before degradation; (right) is the scanning electron microscope diagram of PAM after degradation.

[0027] Figure 6 It is an infrared spectrum diagram of PAM before and after degradation.

[0028] Figure 7 It is a liquid chromatography diagram of acrylamide after degradation. Detailed Description of the Invention

[0029] The present invention will be further described in detail below with reference to the drawings and through specific embodiments.

[0030] Example 1:

[0031] A screening method for a strain used to degrade polyacrylamide in produced water from oilfields, comprising the following steps:

[0032] (1) Collect bauxite mud containing polyacrylamide as the inoculation source. Take the bauxite sludge containing polyacrylamide and add it to the enrichment medium, and incubate it at a constant temperature with shaking for 4 days.

[0033] (2) Inoculate the bacterial suspension obtained in step (1) into the basic liquid medium, so that the polyacrylamide concentration in the medium is successively 100 ppm to 500 ppm.

[0034] (3) Dilute the bacterial suspension obtained in step (2) proportionally and evenly coat it on the Martin's medium, and incubate it at a constant temperature to obtain multiple strains of fungi with different morphologies.

[0035] (4) Inoculate the multiple strains of fungi obtained in step (3) into the Martin's medium by the method of continuous streaking, and incubate them at a constant temperature for purification.

[0036] (5) Prepare spore suspensions from the purified fungi obtained in step (4), inoculate them into the polyacrylamide medium, culture for 8 days, and then measure their respective polyacrylamide degradation abilities to obtain the optimal degrading fungi.

[0037] (6) Add the spore liquid of the optimal PAM-degrading fungi screened in step (5) to medium ① in Table 1 of the acclimation medium, incubate it at a constant temperature with shaking for 4 days, then pour out half of the supernatant and continue to add the same volume of medium ①, and continue to culture under the same conditions for 4 days.

[0038] (7) Pour out the supernatant completely after culturing in step (6), add the bacterial cells to medium ②, and culture according to the methods of steps (6) and (7) until transferred to medium ⑥.

[0039] (8) Inoculate the fungi in medium ⑥ in step (7) onto the Martin's medium. After growing to maturity, obtain the spore suspension of the optimally acclimated fungi according to the method of step (5).

[0040] The compositions of various media in the above screening method are as follows:

[0041] The enrichment medium is made from the following raw materials: peptone 10 g / L and glucose 40 g / L, natural pH.

[0042] The basal liquid medium is made from the following raw materials: glucose 3 g / L, NH4Cl 0.1 g / L, KH2PO4 3 g / L, MgSO4 1 g / L, vitamin B1 8 mg / L, CuSO4·5H2O 8 mg / L, ZnSO4·7H2O 12 mg / L, and PAM 0.5 g / L, with natural pH. The Martin's medium is made from the following raw materials: glucose 10 g / L, peptone 5 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.5 g / L, with natural pH. To 1000 ml of this culture solution, add 3 ml of 1% rose bengal aqueous solution, with natural pH. When in use, add 0.3 ml of 1% streptomycin solution to every 100 ml of the medium.

[0043] Table 1 Composition of the domestication medium

[0044]

[0045] The above media are all wet sterilized at 121°C and 0.1 MPa for 30 min.

[0046] The following analysis is carried out on the screened fungus #12:

[0047] 1. The polyacrylamide-degrading fungus #12 mentioned above is identified by morphological characteristics and ITS sequence analysis, and the results are as follows:

[0048] (1) Morphological characteristics: Referring to the "Fungal Identification Handbook", the polyacrylamide-degrading fungus #12 is inoculated onto the Martin's medium and grows well after 4 days of cultivation at 30°C. The conidia are dark green, clustered and cottony, as Figure 1 shown.

[0049] (2) ITS gene amplification and sequence analysis:

[0050] The internal transcribed spacer ITS of the fungus is PCR amplified using the universal primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4-R (5’-TCCTCCGCTTATTGATATGC-3’) for fungal ITS. The amplification system is (25 µL); amplification conditions: after pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 90 s, for 30 cycles, and then extension at 72°C for 10 min. The PCR amplification product is sequenced by Shanghai Sangon Biotech Co., Ltd., and finally the measured ITS sequence is compared and analyzed through the NCBI database BLAST. Sequence analysis is carried out using MEGA 11 software to construct a phylogenetic tree (see Figure 2 ).

[0051] The above sequencing results showed that the sequence length of the polyacrylamide-degrading fungus #12 was 577 bp. The sequencing results were subjected to BLAST homology alignment in NCBI. The polyacrylamide-degrading fungus #12 was BLAST-aligned with its ITS sequence in NCBI, and it was found that the polyacrylamide-degrading fungus had the highest similarity with Trichoderma asperellum, with a similarity of 99.61%. In summary, the polyacrylamide-degrading fungus #12 was identified as Trichoderma asperellum.

[0052] 2. Comparison of the degradation rates of the polyacrylamide-degrading fungus before and after domestication

[0053] The fungus #12 before and after domestication was respectively prepared into spore suspensions, added to the basal liquid medium, and cultured for 8 days, and then the degradation rates were compared.

[0054] The degradation rates of the fungus #12 on PAM before and after domestication are as Figure 3 shown. The results showed that the degradation rate of the fungus #12 on PAM increased by 23.45% after domestication.

[0055] 3. Optimization of the degradation conditions of the polyacrylamide-degrading fungus

[0056] The fungus #12 was prepared into a spore suspension. The inoculum amount, pH, and temperature during the degradation of PAM by the fungus #12 were changed, and the degradation effect was analyzed.

[0057] The degradation results of the fungus #12 under different conditions are as Figure 4 shown. The results showed that when the inoculum amount was adjusted between 1% and 6%, the optimal inoculum amount was 5%; when the pH was adjusted between 4 and 8, the optimal pH was 5; when the temperature was adjusted between 25 °C and 45 °C, the optimal temperature was 30 °C.

[0058] Example 2: Degradation verification experiment

[0059] 1. Degradation of polyacrylamide solution by the fungus #12:

[0060] The fungus #12 was prepared into a spore suspension. Within the experimental condition range, the optimal degradation conditions of the fungus #12 were: pH 5, inoculum amount 5%, and temperature 30 °C. Under the above conditions, the domesticated fungus #12 was added to the basal liquid medium and cultured for 8 days. The degradation rate of the fungus #12 on PAM and the viscosity reduction rate of the PAM molecular weight were 64.18% and 88.03% respectively.

[0061] 2. Changes in the structure and functional groups of polyacrylamide before and after degradation by the fungus #12

[0062] The PAM before and after degradation by the fungus #12 was characterized and verified, and the imaging results were obtained by using a scanning electron microscope (SEM) asFigure 5 As shown in the figure, the surface of the PAM sample is very compact and shiny. After fungal degradation, the structure of PAM is significantly damaged, with a large number of cavities, cracks and depressions appearing. Fungal hyphae attach to the surface of PAM or extend into the degraded pores, forming a structure similar to a biofilm. It shows that fungus #12 can not only catalyze the degradation of PAM, but also the hyphae produced during its growth can physically damage the surface structure of PAM, break the PAM structure, and generate small molecule substances.

[0063] In-situ infrared spectroscopy (In-Situ IR) was used to determine the changes in functional groups before and after PAM degradation. The changes in functional groups of fungus #12 before and after degradation are as Figure 6 shown. After degradation, the stretching vibration peak of N-H at 3433 cm -1 becomes sharper, and at the same time, the in-plane bending vibration peak of N-H at 1607 cm -1 weakens, indicating that a large amount of amide groups in the side chain of PAM are hydrolyzed, but not completely. 2969 cm -1 is the characteristic absorption peak of the asymmetric stretching vibration of methyl. This peak becomes stronger and blueshifts after degradation, indicating that the long-chain molecules of PAM after degradation are decomposed into small molecule substances, resulting in an increase in the number of terminal methyl groups. 1700 - 1630 cm -1 is the stretching vibration peak of C=O. The stretching vibration absorption band of C=O becomes sharper after degradation, and the transmittance decreases, indicating that more C=O bonds are generated after degradation, and ketones or carboxylic acids appear. 1200 - 1000 cm -1 is the stretching vibration peak of C-O and the stretching vibration of C-C. The 1048 cm -1 appearing after degradation may be the generation of alcohols.

[0064] High performance liquid chromatography (HPLC) was used to determine whether acrylamide monomer exists in the degradation solution after degradation. The concentration change of acrylamide in the degradation solution after fungus #12 degradation is as Figure 7 shown. Acrylamide monomer was not detected in the degradation solution after degradation.

[0065] The above experiments illustrate that the fungus #12 provided by the present invention can effectively degrade polyacrylamide.

Claims

1. A polyacrylamide-degrading fungus, characterized in that: The strain is deposited in the China General Microbiological Culture Collection Center (CGMCC) with the accession number: CGMCC No. 41818, the deposit date: February 24, 2025, the deposit address: Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, Postal Code: 100101, the classification name is Trichoderma asperelloides, and the strain number is #12.

2. The polyacrylamide-degrading fungus according to claim 1, characterized in that: The ITS sequence of the strain is as described in SEQ ID NO.

1. Through BLAST comparison, it was found that #12 had at least 99% similarity with the gene sequence of Trichoderma acanthosporum.

3. The polyacrylamide-degrading bacteria according to claim 1, characterized in that: The polyacrylamide-degrading fungus can be used to degrade polyacrylamide; the polyacrylamide is non-ionic polyacrylamide.

4. A method for screening and acclimating polyacrylamide-degrading bacteria, characterized in that: The steps include: (1) Collect bauxite sludge containing polyacrylamide as the inoculation source, add the bauxite sludge containing polyacrylamide into the enrichment medium, and culture it at a constant temperature with shaking for 4 days; (2) inoculating the bacterial suspension obtained in step (1) into a basic liquid culture medium so that the concentration of polyacrylamide in the culture medium is 100 ppm to 500 ppm; (3) diluting the bacterial suspension obtained in step (2) in proportion and evenly applying it to Martin's medium, and culturing at a constant temperature to obtain multiple fungal strains with different morphologies; (4) inoculating the multiple fungal strains obtained in step (3) into Martin's medium by continuous streaking method, and culturing them at a constant temperature for purification; (5) preparing spore suspensions of the purified fungi obtained in step (4) and inoculating them into polyacrylamide culture medium, and then determining their respective polyacrylamide degradation abilities after culturing for 8 days to obtain the optimal degradation fungi; (6) adding the spore liquid of the optimal PAM-degrading fungus selected in step (5) to medium No. ① of the acclimation medium, culturing with constant temperature shaking for 4 days, pouring out half of the supernatant, and then adding the same volume of medium No. ①, and continuing to culture under the same conditions for 4 days; (7) After the supernatant after the culture in step (6) is completely poured out, it is added to medium No. ②, and cultured according to the methods of steps (6) and (7) until it is transferred to medium No. ⑥; (8) The fungi in medium No. ⑥ in step (7) are inoculated onto Martin's medium, and after they grow to maturity, a spore suspension of the best domesticated fungi is obtained according to the method of step (5).

5. Application of polyacrylamide-degrading fungi in the treatment of PAM-containing sludge.

6. The use according to claim 5, characterized in that When the polyacrylamide-degrading fungus degrades polyacrylamide, its inoculation amount is between 1% and 6%, the pH is between 4 and 8, and the temperature is between 25° C. and 45° C.

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