A Cladosporium fungus and its application

Abstract

The present invention relates to the field of microbial technology, and in particular to a Cladosporium fungus and its use. The present invention provides a Cladosporium sp. fungus DLSH-DA-1, which is deposited in the General Microbiology Center of the China Culture Collection Administration Committee with a deposit number of CGMCC No. 19928. The strain has excellent salt tolerance, especially tolerance to high concentrations of sulfate, and has the ability to efficiently remove COD from wastewater in a high-salt environment. It is suitable for removing environmentally harmful substances such as COD from high-salt wastewater, especially high-sulfate wastewater.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a Cladosporium fungus and application thereof. Background Art

[0002] Wastewater generated by industries such as organic chemicals, food processing, and microbial fermentation contains not only high concentrations of organic residues but also a large amount of inorganic salt ions, such as Cl － 、SO4 2- 、Na + In some wastewater produced by organic acid fermentation, sulfate concentrations can reach as high as 30,000–50,000 mg / L, and COD can exceed 10,000 mg / L. The abundant sulfate ions make this high-salinity wastewater difficult to biodegrade. Furthermore, due to its high organic content, physicochemical processes such as membrane separation and concentration crystallization are neither economical nor desirable. Therefore, screening for strains that can tolerate high-salinity industrial wastewater environments is crucial for its treatment. Summary of the Invention

[0003] The purpose of the present invention is to provide a Cladosporium fungus and application thereof.

[0004] The key to using microorganisms to treat saline wastewater lies in their tolerance to high-salinity environments and their ability to remove organic matter from wastewater under these conditions. This study provides a Cladosporium sp. fungus strain, DLSH-DA-1, that can rapidly adapt to the high salt ion concentrations found in wastewater and effectively remove most organic matter and some inorganic pollutants.

[0005] The Cladosporium sp. fungus DLSH-DA-1 was deposited on July 13, 2020 at the General Microbiology Center of the China Culture Collection Administration (CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), and was classified as Cladosporium sp. with the deposit number CGMCC No. 19928.

[0006] The solid culture characteristics of strain DLSH-DA-1 are: culture on potato dextrose agar (PDA) medium, culture temperature is 28±1℃, culture for 5 to 7 days, morphology is: colony diameter is 29 to 32 mm, brown-green, flat surface, velvety texture, indigo blue on the back, no exudate, and no soluble pigment.

[0007] The microscopic morphological characteristics of strain DLSH-DA-1 are as follows: conidiophores occur on hyphae, are light brown, spore-bearing cells are terminal or lateral, and the spore stalks are separate, upright or slightly curved; conidia are chain-like, branched conidia are columnar or fusiform, measuring 7-21×2.5-5μm, and terminal conidia are fusiform or elliptical, measuring 1.5-6.5×2.5-3.5μm; no thick-walled spores were observed.

[0008] The ITS rDNA gene sequence of strain DLSH-DA-1 is shown in SEQ ID NO. 1. Based on the results of sequence alignment of the morphological characteristics of strain DLSH-DA-1 and its ITS sequence in professional databases based on the NCBI database, the strain was identified as a potential new species of Cladosporium sp.

[0009] Based on the strain DLSH-DA-1, the present invention provides a bacterial agent containing the Cladosporium sp. fungus DLSH-DA-1.

[0010] The present invention does not particularly limit the type of the above-mentioned microbial agent, which can be a solid or liquid microbial agent. In addition to the Cladosporium sp. fungus DLSH-DA-1, the microbial agent may also contain a culture medium for culturing the DLSH-DA-1 strain, metabolites of the DLSH-DA-1 strain, and carriers or other excipients permitted in the field of microbial preparations.

[0011] The present invention also provides a method for preparing the bacterial agent, which comprises the following steps:

[0012] (1) Cladosporium sp. fungus DLSH-DA-1 was inoculated into potato dextrose agar medium containing 2-4 g / L sulfate and activated and cultured at 25-30°C for 48-72 h;

[0013] (2) inoculating the activated Cladosporium sp. fungus DLSH-DA-1 in step (1) into a potato glucose liquid culture medium containing 1000-15000 mg / L sulfate, and shaking culture at 25-30° C. for 72-120 hours to obtain a culture solution;

[0014] (3) The culture solution obtained in step (2) is prepared into a solid or liquid bacterial agent.

[0015] The sulfate mentioned above may be Na2SO4.

[0016] The present invention also provides a method for culturing the Cladosporium sp. fungus DLSH-DA-1, comprising the following steps:

[0017] (1) Strain activation: The strain DLSH-DA-1 was inoculated into potato dextrose agar (PDA) medium containing 2-4 g / L sulfate by streaking and cultured at 25-30°C for 48-72 h;

[0018] (2) Liquid culture: After the activation of the strain is completed, the larger activated colonies on the plate are selected, and the planar spores are picked out and inoculated into a potato glucose liquid medium containing 1000-15000 mg / L sulfate. The culture is shaken at 25-30°C and 100-200 rpm for 72-120 h to obtain the culture medium of DLSH-DA-1.

[0019] The sulfate mentioned above may be Na2SO4.

[0020] The invention provides a sewage treatment agent, which contains the Cladosporium sp. fungus DLSH-DA-1 or the bacterial agent.

[0021] Based on the function of the strain DLSH-DA-1 provided by the present invention, the present invention provides the use of the Cladosporium sp. fungus DLSH-DA-1 or the bacterial agent or the sewage treatment agent in wastewater or sewage treatment.

[0022] The present invention provides use of Cladosporium sp. fungus DLSH-DA-1 or the bacterial agent or the sewage treatment agent in high-salt wastewater or sewage treatment.

[0023] Preferably, the high-salt wastewater or sewage is wastewater or sewage with a high sulfate content. The sulfate content in the wastewater or sewage is preferably 800-9200 ppm.

[0024] The present invention provides application of Cladosporium sp. fungus DLSH-DA-1 or the bacterial agent or the sewage treatment agent in treating dibasic acid fermentation wastewater.

[0025] Wastewater from dibasic acid fermentation, a typical example, contains some fermentation broth nutrients, high COD, and high sulfate levels. The sulfate is derived from sulfuric acid added during acid precipitation of the fermentation broth. Using strain DLSH-DA-1 to biologically purify difficult-to-treat residual organic matter in this wastewater not only effectively removes COD and other harmful substances, resolving the issue of excessively high COD, but also provides a clean treatment environment for subsequent sulfate resource utilization, significantly reducing the pressure on subsequent sulfate treatment processes. Using this strain to treat dibasic acid fermentation wastewater ultimately enables the wastewater to meet discharge standards.

[0026] The present invention also provides use of the Cladosporium sp. fungus DLSH-DA-1 or the bacterial agent or the sewage treatment agent in removing COD from wastewater or sewage.

[0027] The invention provides a method for treating wastewater or sewage. The method comprises: inoculating Cladosporium sp. fungus DLSH-DA-1 into wastewater or sewage to be treated for culturing.

[0028] Preferably, the culture temperature is 25-30°C.

[0029] Before inoculating wastewater or sewage, the Cladosporium sp. fungus DLSH-DA-1 is preferably first inoculated into a sulfate-containing potato dextrose medium at 25-30° C. for seed culture to obtain seed liquid, which is then inoculated into the wastewater or sewage to be treated.

[0030] The present invention has the following beneficial effects: A highly salt-tolerant strain, DLSH-DA-1, was obtained through screening and cultivation. This strain exhibits excellent salt tolerance, particularly high sulfate concentrations (up to 9000 mg / L), and is capable of efficiently removing COD from wastewater in high-salt environments. Compared to existing highly salt-tolerant strains, this strain is more suitable for removing environmentally harmful substances such as COD from high-salt wastewater, especially high-sulfate wastewater.

[0031] In addition, the strain DLSH-DA-1 has high stability after being cultured in a solid culture medium containing sulfate. It can be stored under sterile conditions at room temperature for 30 to 45 days and stored at 0 to 4°C for 3 to 5 months without inactivation. Its spore activity will not be significantly reduced, making it easy to store. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a microscopic morphology of the highly salt-tolerant strain DLSH-DA-1 in Example 1 of the present invention under a microscope.

[0033] Figure 2This is a colony morphology diagram of the highly salt-tolerant strain DLSH-DA-1 on solid culture medium in Example 1 of the present invention.

[0034] Figure 3 This is the phylogenetic tree of ITS rDNA of the highly salt-tolerant strain DLSH-DA-1 in Example 1 of the present invention.

[0035] Figure 4 This is a phylogenetic tree of the Actin gene of the highly salt-tolerant strain DLSH-DA-1 in Example 1 of the present invention.

[0036] Figure 5 This is the growth curve of the highly salt-tolerant strain DLSH-DA-1 in Example 3 of the present invention under actual sewage salt concentration.

[0037] Figure 6 This is the COD removal rate of the highly salt-tolerant strain DLSH-DA-1 in Example 3 of the present invention under actual sewage salt concentration. DETAILED DESCRIPTION

[0038] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0039] Example 1 Screening and identification of highly salt-tolerant strain DLSH-DA-1

[0040] The screening process of the highly salt-tolerant strain DLSH-DA-1 provided by the present invention is mainly as follows:

[0041] (1) Sampling: Wild strains grown in the wastewater of long-chain dicarboxylic acid fermentation in the wastewater treatment pool of the Key Laboratory of Biomass Catalytic Conversion of Dalian Research Institute of Petrochemical Technology were collected. The wastewater data are shown in Table 1.

[0042] Table 1 Data of wastewater from dibasic acid fermentation

[0043]

[0044]

[0045] (2) Strain activation: Take PDA culture medium powder and pure water, add a small amount of sewage to pH 5.0, prepare PDA solid culture medium, sterilize and place at room temperature, perform streak inoculation, the strain can grow rapidly, generally within 1 to 2 days, the colony diameter exceeds 10 mm, that is, the activation is complete.

[0046] (3) Initial acclimation: Take 20g of PDA medium powder, 800g of pure water, and 200g of high-salt wastewater, mix them to a pH of about 4.0, add NaOH to adjust the pH to 4.5, sterilize and prepare plates, and cool to room temperature. Select spores with larger colony diameters from the activated plates in step (2) for streak inoculation. After culturing in a sterile room for 3-5 days, select colonies with a diameter of ≥20mm, select spores for streak inoculation, and repeat 10-15 times until the strain is fully adapted to the culture conditions and then enter the next stage of acclimation culture.

[0047] (4) Secondary domestication: After the initial domestication, the surface spores with larger colony diameters in the 15th batch of plates were taken and transferred to a solid culture medium with a high-salt sewage ratio of 30% and a pH of 4.25. This was repeated 10 to 15 times until the strain was fully adapted to this culture condition. The high-salt sewage was then increased and the pH value was decreased gradually until the sewage ratio reached 100% and the pH was 3.5. Domestication was continued in this manner.

[0048] A strain was screened by the above method and named DLSH-DA-1. The morphology of the strain under the microscope is as follows: Figure 1 As shown in the figure, the details are as follows: conidiophores are light brown and grow on hyphae. Conidiophores are terminal or lateral, and the spore-producing cells are separated by stalks, erect or slightly curved. Conidia are chain-like, branched conidia are columnar or fusiform, and are 7-21×2.5-5μm in size. Terminal conidia are fusiform or elliptical, and are 1.5-6.5×2.5-3.5μm in size. No thick-walled spores are observed. The morphology of solid culture is as follows: Figure 2 As shown, the details are as follows: on PDA medium, cultured at 25°C for 7 days, the colony diameter is 29-32 mm, brown-green; the surface is flat; the texture is velvety; the reverse side is indigo blue; no exudate is produced; no soluble pigment is produced.

[0049] Sequencing analysis revealed the ITS rDNA sequence of strain DLSH-DA-1 as shown in SEQ ID NO. 1. Sequence alignment of the ITS rDNA with specialized databases based on the NCBI database revealed sequence similarities between the ITS rDNA of strain DLSH-DA-1 and those of related species, as shown in Table 2.

[0050] Table 2 ITS rDNA sequence alignment results

[0051] Related species Sequence similarity Cladosporium anthropophilum CBS 140685T(LN834437) 100.0％ <![CDATA[Cladosporium austroafricanum CBS 140482 T (KT600381)]]> 99.8％ <![CDATA[Cladosporium phaenocomae CBS 128769 T (NR_119950)]]> 99.8％ <![CDATA[Cladosporium funiculosum CBS 122129 T (HM148094)]]> 99.8％ <![CDATA[Cladosporium montecillanum CBS 140486 T (KT600406)]]> 99.8％ <![CDATA[Cladosporium australiense CPC 13226 T (NR_119837)]]> 99.8％ <![CDATA[Cladosporium angustisporum CPC 12437 T (NR_111530)]]> 99.8％ <![CDATA[Cladosporium gamsianum CPC 11807 T (NR_111533)]]> 99.8％ <![CDATA[Cladosporium ipereniae CBS 140483 T (KT600394)]]> 99.6％ <![CDATA[Cladosporium varians CBS126362 T (HM148224)]]> 99.6％ <![CDATA[Cladosporium myrtacearum CBS 140483 T (NR_119849)]]> 99.6％ Cladosporium asperulatum CPC 14040(NR_119836) 99.6％ <![CDATA[Cladosporium pini-ponderosae CBS 124456 T (FJ936160)]]> 99.6％

[0052] The MEGA5.0 software was used to display the phylogenetic tree of ITS rDNA sequences of strain DLSH-DA-1 and related species using the proximity joining method ( Figure 3), the similarity calculation was repeated 1000 times, and the developmental tree nodes in the figure only show the values ​​with bootstrap values ​​greater than 70%. The superscript "T" represents the type strain, and SHDA-1 represents the strain DLSH-DA-1.

[0053] Sequencing analysis revealed that the Actin gene sequence of strain DLSH-DA-1 is shown in SEQ ID NO. 2, which is identical to that of Cladosporium anthropophilum CBS 140685. T The sequence similarity of Actin gene between LN834621 and Cladosporiumcladosporioides CBS 112388T (HM148490) was the highest, both of which were 95.1%.

[0054] The phylogenetic tree of Actin gene sequences of strain DLSH-DA-1 and related species was displayed by the proximity joining method using MEGA5.0 software ( Figure 4 ), the similarity calculation was repeated 1000 times, and the developmental tree nodes in the figure only show the values ​​with bootstrap values ​​greater than 70%. The superscript "T" represents the type strain, and SHDA-1 represents the strain DLSH-DA-1.

[0055] After identification, the strain DLSH-DA-1 belongs to the genus Cladosporium sp. The strain DLSH-DA-1 was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101) on July 13, 2020, and was classified and named Cladosporium sp. with the deposit number CGMCC No. 19928.

[0056] Example 2 Cultivation of strain DLSH-DA-1 and preparation of bacterial solution

[0057] This example provides a method for culturing and preparing a bacterial solution of the strain DLSH-DA-1, as follows:

[0058] (1) Strain activation: The strain DLSH-DA-1 was inoculated into potato dextrose agar (PDA) medium containing 4 g / L sodium sulfate by streaking and cultured at 27°C for 48 h.

[0059] (2) Liquid culture: After the activation of the strain, select the largest activated colony on the plate, pick out the planar spores, and inoculate them into a potato glucose liquid medium containing 4 g / L sodium sulfate. Culture at 28°C and 150 rpm with shaking for 72 h to obtain the DLSH-DA-1 bacterial liquid.

[0060] The preparation method of potato dextrose agar (PDA) medium is as follows: 40 g of PDA dry powder is dissolved in water and the volume is adjusted to 1 L, and then sterilized to prepare a solid culture plate.

[0061] The preparation method of potato glucose liquid culture medium is as follows: 20g potato extract powder, 5g glucose, dissolve in water and make up to 1L, sterilize and cool to room temperature.

[0062] Example 3 Determination of salt tolerance and COD degradation ability of strain DLSH-DA-1

[0063] Prepare liquid culture medium containing wastewater as simulated wastewater: 2g potato extract powder, 1g glucose, 900g pure water, dibasic acid fermentation wastewater (COD value content is about 7400mg / L, SO4 2－ The content of the purified water was about 12000ppm) per 100g, and the pH was 5.2. On this basis, the amount of purified water was continuously reduced to 200g at intervals of 100g, while the amount of sewage was increased to 800g, at which time the pH was 3.3.

[0064] The bacterial solution of strain DLSH-DA-1 (the concentration of live bacteria of DLSH-DA-1 is 0.45 g / L dry weight) was taken and added to a conical flask at a volume ratio of 1:20 with the simulated wastewater. The culture was carried out using a shaking table. During the culture process, the temperature was controlled at 30 °C and the speed was 100 rpm. Samples were taken at regular intervals and the bacterial density (OD) was measured using a spectrophotometer. 600 ), draw the growth curve of the strain, the growth curve of the strain at different salt concentrations is as follows Figure 5 At the same time, the COD value of the final reaction solution was measured to determine the COD removal rate of the strain. After 72 hours of culture, the COD removal rate of the strain at different salt concentrations was as follows: Figure 6 shown.

[0065] The above results show that as the salt concentration increases, the growth of strain DLSH-DA-1 slows down relatively, but after a certain adaptation period, strain DLSH-DA-1 can grow rapidly; at about 9120ppm SO4 2－ Under the environment of 994ppm SO4, the strain DLSH-DA-1 can still maintain a relatively fast growth rate, and the corresponding COD removal rate can still be as high as 75.941%. 2－ Under the above conditions, the COD removal rate of strain DLSH-DA-1 was 93.271%. This shows that strain DLSH-DA-1 has strong salt tolerance and COD removal ability.

[0066] The degradation activity of strain DLSH-DA-1 on inorganic pollutants was further tested by using the above-mentioned diluted 70wt% simulated wastewater as the treatment object, in which SO4 2- The concentration of Na, P and Si was 8007ppm, and the concentrations of Na, P and Si were 436ppm, 126ppm and 46.0ppm respectively. The bacterial solution of strain DLSH-DA-1 (the live bacterial concentration of DLSH-DA-1 was 0.45g / L by dry weight) was taken and added to a conical flask at an inoculum volume ratio of 1:20 with the simulated wastewater. The culture was carried out using a shaking table. During the culture process, the temperature was controlled at 30°C and the speed was 100rpm. After 72h of culture, the contents of Na and P elements were reduced to 344ppm and 115ppm respectively, while the content of Si element after degradation was reduced to below the detection limit (undetectable). The above results show that the strain DLSH-DA-1 also has a certain ability to degrade inorganic pollutants in a high-salt environment.

[0067] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein. Sequence Listing <110> China Petroleum & Chemical Corporation Dalian Research Institute of Petrochemical Industry <120> A Cladosporium fungus and its application <130> KHP211122474.4 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 496 <212> DNA <213> Artificial Sequence <400> 1 cgtaacaagg tctccgtagg tgaacctgcg gagggatcat tacaagtgac cccggtctaa 60 ccaccgggat gttcataacc ctttgttgtc cgactctgtt gcctccgggg cgaccctgcc 120 ttcgggcggg ggctccgggt ggacacttca aactcttgcg taactttgca gtctgagtaa 180 acttaattaa taaattaaaa cttttaacaa cggatctctt ggttctggca tcgatgaaga 240 acgcagcgaa atgcgataag taatgtgaat tgcagaattc agtgaatcat cgaatctttg 300 aacgcacatt gcgccccctg gtattccggg gggcatgcct gttcgagcgt catttcacca 360 ctcaagcctc gcttggtatt gggcatcgcg gtccgccgcg tgcctcaaat cgaccggctg 420 ggtcttctgt cccctaagcg ttgtggaaac tattcgctaa agggtgttcg ggaggctacg 480 ccgtaaaaca acccca 496 <210> 2 <211> 223 <212> DNA <213> Artificial Sequence <400> 2 aggccggttt cgccggtgac gatgcgccca gagccgtttt ccgtaagtct gaagacacct 60 gtttcgcccg tctcgcaatt ccgagctgac acccctccca gcttccattg tcggcagacc 120 ccgtcaccat gggtatgcat tctccccgcg agcctccctg tcgcgcgcag ccaatctaac 180 ccctcggcag tatcatgatc ggtatgggcc agaaggactc gta 223

Claims

1. A Cladosporium sp. fungus DLSH-DA-1, characterized in that: It is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number CGMCC No.19928.

2. A bacterial agent, characterized in that The invention also contains the Cladosporium sp. fungus DLSH-DA-1 according to claim 1 .

3. The method for preparing the microbial agent according to claim 2, characterized in that: The steps include: (1) Cladosporium sp. fungus DLSH-DA-1 was inoculated into potato dextrose agar medium containing 2-4 g / L sulfate and activated and cultured at 25-30°C for 48-72 h; (2) inoculating the activated Cladosporium sp. fungus DLSH-DA-1 in step (1) into a potato glucose liquid culture medium containing 1000-15000 mg / L sulfate, and shaking culture at 25-30° C. for 72-120 hours to obtain a culture solution; (3) The culture solution obtained in step (2) is prepared into a solid or liquid bacterial agent.

4. A sewage treatment agent, characterized in that The invention contains the Cladosporium sp. fungus strain DLSH-DA-1 according to claim 1 or the bacterial agent according to claim 2 .

5. Use of the Cladosporium sp. fungus DLSH-DA-1 according to claim 1, the bacterial agent according to claim 2, or the sewage treatment agent according to claim 4 in the treatment of high-sulfate wastewater.

6. Use of the Cladosporium sp. fungus DLSH-DA-1 according to claim 1, the bacterial agent according to claim 2, or the sewage treatment agent according to claim 4 in the treatment of dibasic acid fermentation wastewater.

7. Use of the Cladosporium sp. fungus DLSH-DA-1 according to claim 1, the bacterial agent according to claim 2, or the sewage treatment agent according to claim 4 in removing COD from high-sulfate wastewater.

8. A method for treating high-sulfate wastewater, characterized in that: include: The Cladosporium sp. fungus DLSH-DA-1 described in claim 1 is inoculated into the high-sulfate wastewater to be treated for cultivation.

9. The method according to claim 8, characterized in that The culture temperature is 25-30°C.

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