Cronobacter sp. And application thereof in PTA (purified terephthalic acid) wastewater degradation

Abstract

The invention discloses Cronobacter sp. And application thereof in PTA (purified terephthalic acid) wastewater degradation, and belongs to the technical field of biodegradation. In order to screen out microorganisms capable of efficiently degrading COD (Chemical Oxygen Demand) in PTA (Purified Terephthalic Acid) production wastewater, a strain of Cronobacter malonaticus H4 is separated from sludge of a Weizhou sewage treatment plant, the strain can normally grow under the alkaline condition that the pH (Potential of Hydrogen) is 7.0 to 11.0 and can degrade COD in the PTA wastewater, and the COD degradation rate of the PTA wastewater with the COD content of 90000 mg / L to 100000 mg / L can reach 76.73 percent. The method for degrading the COD in the PTA wastewater by using the cronobacter malonate H4, provided by the invention, has the characteristics of simple process, low requirement on the process, safety and environment friendliness.

Description

Technical Field

[0001] The present invention belongs to the technical field of biodegradation, and specifically relates to a strain of Cronobacter and its application in degrading PTA wastewater. Background Art

[0002] Purified Terephthalic Acid (PTA) is a type of chemical widely used in industries such as chemical engineering, electronics, and construction. It is generally obtained from p-xylene through a series of chemical reactions, and its preparation can be divided into two steps: oxidizing p-xylene into crude xylene and purifying the crude xylene.

[0003] As the main producer of PTA, China's PTA output increased by 16.3% year-on-year in 2021, reaching 66.3 million tons. And for every ton of PTA produced, 3 - 4 cubic meters of sewage is generated. The organic sewage generated during the PTA production process mainly consists of Terephthalic Acid (TA), p-toluic Acid (PT), Benzoic acid (BA), ethyl acetate, etc. At the same time, during the PTA production process, cobalt acetate / manganese, etc. are often used as catalysts in the reaction system. Therefore, some Co 2+ , Mn 2+ .

[0004] PTA production wastewater has the following characteristics:

[0005] (1) There are large fluctuations in the quality and quantity of the wastewater. Under conventional process conditions, PTA production wastewater contains a large amount of reactive residual substances, resulting in significant changes in the concentration of various impurities. At the same time, regular cleaning of pipelines, large-scale maintenance of equipment, etc. will all cause changes in water quality and quantity.

[0006] (2) High Chemical Oxygen Demand (COD). The main source of COD in PTA production wastewater is various refractory aromatic organic compounds, and the concentration of COD fluctuates between 4000 - 21000 mg / L.

[0007] (3) The discharged wastewater has the characteristic of high temperature. Since in the PTA process, the operating temperature of the oxidation tank is generally above 150°C, the temperature of the discharged water is above 45°C, and some can even reach above 80°C. Therefore, the wastewater must go through a series of cooling processes before it can be sent into the biochemical treatment unit.

[0008] (4) The pH fluctuates greatly. In the conventional industrial production process, since a lot of acetic acid is used as a solvent, the pH of most wastewater is very low. However, during the process of cleaning the device, strong alkali with a relatively high pH value must be added to neutralize it. Therefore, the pH of PTA production wastewater usually varies within the range of 3 - 12.

[0009] Compared with physical and chemical methods, the microbial method can effectively remove COD in PTA production wastewater, efficiently and stably improve the water quality to within the national discharge standards. At the same time, its operation is flexible, and it can be adjusted and optimized according to the actual pollution source situation, and the operation is relatively simple. Moreover, almost no chemicals need to be used, which will not cause secondary pollution, has environmental protection, can reduce the pollution of wastewater discharge to the surrounding environment, and reduce the damage to natural resources. However, at present, there are still problems in using the biological method to degrade COD in PTA production wastewater, such as excessive requirements for water quality and environment, fewer types of microorganisms that meet the requirements, low degradation efficiency, and inability to be widely applied in industrial production. Therefore, if strains that can efficiently degrade COD in PTA production wastewater can be screened, it will provide strain resources for the microbial degradation of PTA production wastewater. Summary of the Invention

[0010] In order to screen microorganisms that can efficiently degrade COD in PTA production wastewater and use them for the degradation of PTA production wastewater, the present invention screened a strain of bacteria H4 from the sludge of Changzhou Sewage Treatment Plant. After identification, it was found that this strain was Cronobacter malonaticus, and it was found that adding the cells of this strain to PTA wastewater could achieve the effect of efficiently degrading COD in PTA wastewater.

[0011] To solve the above technical problems and achieve the corresponding technical effects, the present invention specifically provides the following technical solutions:

[0012] The first object of the present invention is to provide a strain of Cronobacter malonaticus H4, which is preserved in the China General Microbiological Culture Collection Center, with the preservation number of CGMCC No. 29958, the preservation date of March 7, 2024, and the address of the preservation unit is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0013] The second object of the present invention is to provide the application of the above-mentioned Cronobacter malonaticus H4 in degrading COD in PTA wastewater.

[0014] The third object of the present invention is to provide the application of the above-mentioned Cronobacter malonaticus H4 in the preparation of a microbial preparation for degrading COD in PTA wastewater.

[0015] The fourth object of the present invention is to provide a microbial preparation containing the above-mentioned Cronobacter malonaticus H4.

[0016] The fifth object of the present invention is to provide the application of the above microbial preparation in degrading the COD of PTA wastewater.

[0017] The sixth object of the present invention is to provide the application of the above-mentioned Cronobacter malonaticus H4 or the above microbial preparation in preparing a COD treatment agent for PTA wastewater.

[0018] The seventh object of the present invention is to provide a method for degrading the COD of PTA wastewater, which is to inoculate the above-mentioned Cronobacter malonaticus H4 into a liquid medium, cultivate it until the logarithmic growth phase to obtain a bacterial liquid, and add the bacterial cells obtained after centrifuging the bacterial liquid to the PTA wastewater for treatment.

[0019] In one embodiment of the present invention, the cultivation is carried out in a constant temperature shaker with a pH of 7-11, a temperature of 25-40 °C, and a rotation speed of 100-180 rpm.

[0020] In one embodiment of the present invention, the treatment is carried out under the conditions of a pH of 7-11, a temperature of 25-40 °C, and a rotation speed of 100-180 rpm for 72 h.

[0021] In one embodiment of the present invention, the treatment conditions are a pH of 9.0, a temperature of 30 °C, and a rotation speed of 180 rpm.

[0022] In one embodiment of the present invention, the concentration of the above-mentioned Cronobacter malonaticus H4 in the PTA wastewater is ≥ 2.0×10 9 CFU / mL.

[0023] Advantages of the present invention:

[0024] A strain of Cronobacter malonaticus H4 was isolated from the sludge of Changzhou Wastewater Treatment Plant in the present invention. This strain can grow normally under alkaline conditions with a pH of 7.0-11.0, and can degrade the COD in PTA wastewater. The COD degradation rate of PTA wastewater with a COD content of 90000 mg / L - 100000 mg / L can reach 76.73%. The method for degrading the COD of PTA wastewater provided by the present invention has a simple process, low requirements for the process, and the characteristics of safety and environmental protection. Description of the Drawings

[0025] Figure 1 It is a colony characteristic diagram of Cronobacter malonaticus H4. Detailed Embodiments

[0026] The following will further elaborate on the present invention in combination with specific embodiments and accompanying drawings. The following embodiments facilitate a better understanding of the present invention, but are not used to limit the present invention. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The drugs, reagents, and materials used in the following embodiments can all be obtained through commercial channels unless otherwise specified.

[0027] The PTA wastewater in the following embodiments: The production wastewater from a certain factory in Changzhou contains a large amount of organic and inorganic pollutants, such as heavy metals, salts, etc. The organic pollutants in the PTA wastewater are acetic acid, benzoic acid, terephthalic acid, phthalic acid, isophthalic acid, p-toluic acid, etc. The concentration of acetic acid is 500 - 2000 mg / L, the concentration of benzoic acid is 200 - 500 mg / L, the concentration of terephthalic acid is 1000 - 2500 mg / L, the concentration of phthalic acid is 200 - 100 mg / L, the concentration of isophthalic acid is 20 - 100 mg / L, the concentration of p-toluic acid is 400 - 1000 mg / L. The main inorganic pollutants are acetic acid, cobalt and manganese heavy metals. The concentration of cobalt and manganese metal ions in the wastewater reaches more than 1000 mg / L. Acetic acid is used as a catalyst, and its content fluctuates greatly. The acidic pH is 3 - 4. After adjusting the pH during the production process, the pH value is 8 - 9. The PTA wastewater has the characteristics of complex composition, high chromaticity, and containing toxic and harmful substances.

[0028] The culture medium related to the present invention is as follows:

[0029] LB liquid culture medium: 5 g / L of yeast extract, 10 g / L of tryptone, 10 g / L of sodium chloride, and the balance is distilled water, with a pH of 7.0.

[0030] LB solid culture medium: 5 g / L of yeast extract, 10 g / L of tryptone, 10 g / L of sodium chloride, 20 g / L of agar, and the balance is distilled water, with a pH of 7.0.

[0031] pH adjustment of LB liquid culture medium and LB solid culture medium: According to needs, adjust the pH of LB liquid culture medium or LB solid culture medium to 5.0, 7.0, 9.0, and 11.0 with 5 mol / L sodium hydroxide solution and 0.1 mol / L hydrochloric acid solution.

[0032] The calculation method for the degradation rate of COD concentration in the PTA wastewater involved in the following embodiments:

[0033] Use the rapid digestion spectrophotometry method (HJ / T 399 - 2007) to measure the COD concentration content in the PTA wastewater.

[0034] The calculation formula for the degradation rate of COD concentration in the PTA wastewater is as follows:

[0035] PTA wastewater COD concentration degradation rate = (Initial COD concentration value of PTA wastewater before degradation - COD concentration value of PTA wastewater after degradation) / Initial COD concentration value of PTA wastewater before degradation

[0036] Example 1: Screening and identification of Cronobacter malonate H4

[0037] 1. Screening of strain H4

[0038] (1) Take 10 mL of sludge (density is 0.027 g·mL -1 , pH is 7.66) from Changzhou Sewage Treatment Plant, put it into 90 mL of LB liquid medium, and culture it in an environment of 150 - 180 rpm and 32 °C for 4 - 6 d;

[0039] (2) Take the bacterial liquid obtained in step (1) and inoculate it into a new 100 mL LB liquid medium at an inoculation amount of 5 - 10% by volume, and culture it in an environment of 150 - 180 rpm and 32 °C for 4 - 6 d;

[0040] (3) Take the bacterial liquid obtained in step (2) and inoculate it into a new 100 mL LB liquid medium at an inoculation amount of 5 - 10% by volume, and culture it in an environment of 150 - 180 rpm and 32 °C for 4 - 6 d;

[0041] (4) Take 5 μL of the bacterial liquid obtained in step (3) and add it to the sterilized LB liquid medium, culture it in a shaker at 150 - 180 rpm and 32 °C for 4 - 6 d, then use a pipette tip to aspirate 100 μL of the liquid and spread it on the LB solid medium;

[0042] (5) Place the LB solid medium coated with the bacterial liquid in an incubator at 30 °C for 1 - 2 d, observe the colony morphology, pick a small amount of bacteria from each colony and inoculate them into the LB liquid medium respectively, after culturing for 4 - 6 d, spread the bacterial liquid on the LB solid medium;

[0043] (6) Repeat step (5) multiple times until a single strain is obtained in each LB solid medium;

[0044] (7) Pick a single colony from the medium with a single strain grown, inoculate it into a new 100 mL LB liquid medium, culture it in an environment of 150 - 180 rpm and 32 °C for 4 - 6 d, measure the OD 600 value, when the OD 600 value reaches 0.8, the required bacterial liquid is obtained, and this strain is named H4. Preserve the purified strain H4 in a -80 °C refrigerator for standby.

[0045] 2. Identification of strain H4

[0046] (1) Morphological identification of strain H4

[0047] The strain H4 was inoculated on LB solid medium and cultured upside down in a constant temperature incubator at 30 °C for 7 days. It was observed that the colonies of strain H4 were transparent and colorless, round in shape, and had no protrusions around them (see Figure 1 ).

[0048] (2) Molecular biological identification of strain H4

[0049] Strain H4 was identified as Cronobacter malonaticus by Sangon Biotech (Shanghai) Co., Ltd. through 16S rRNA sequencing. The specific identification method is as follows:

[0050] Genomic DNA of strain H4 was extracted. Using the genomic DNA as a template and universal primers 27F (SEQ ID NO.2) and 1492R (SEQ ID NO.3) as PCR primers for PCR amplification (the PCR reaction system is shown in Table 1, and the PCR reaction program is shown in Table 2). After the PCR products were detected by agarose gel electrophoresis, they were sequenced. The sequence obtained by sequencing (SEQ ID NO.1) was analyzed and compared using the BLAST program (http: / / www.ncbi.nlm.nih.gov / BLAST / ) in NCBI. It was found that the 16S rDNA sequence of strain H4 was most similar to the 16S rDNA sequence of Cronobacter malonaticus, with a similarity of 96.24%. Combining the morphological and molecular biological identification results of strain H4, strain H4 was identified as Cronobacter malonaticus and was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on March 7, 2024. The deposit number is CGMCC No. 29958, and the address of the deposit unit is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0051] Table 1 PCR reaction system

[0052]

[0053] Table 2 PCR reaction program

[0054]

[0055] SEQ ID NO.1:

[0056]

[0057] SEQ ID NO.2: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0058] SEQ ID NO.3: 5'-TACCTTGTTACGACTT-3'.

[0059] Example 2: Optimization of the culture conditions of Cronobacter malonaticus H4

[0060] (1) Optimization of the pH value for the culture of Cronobacter malonaticus H4

[0061] Cronobacter malonaticus H4 was inoculated into LB liquid media with different initial pH values at a volume ratio of 2% respectively. The initial pH values of the LB liquid media were 7.0, 9.0, and 11.0 respectively. The culture was shaken at a temperature of 30 °C and a shaker speed of 180 rpm for 72 h. The OD of the bacterial solution obtained from the media with different initial pH values was measured every 6 h. 600 value.

[0062] As shown in Table 3, Cronobacter malonaticus H4 could grow well in the media with a pH range of 7.0 - 11.0, and its pH tolerance range was 7.0 - 11.0. It entered the logarithmic growth phase earlier under the condition of an initial pH of 9.0. Therefore, its more suitable initial pH was 9.0.

[0063] Table 3 OD of the bacterial solution of Cronobacter malonaticus H4 obtained at different initial pH values 600 value

[0064]

[0065]

[0066] (2) Optimization of the shaker speed for the culture of Cronobacter malonaticus H4

[0067] Cronobacter malonaticus H4 was inoculated into LB liquid media at a volume ratio of 2% respectively. Under the conditions of a pH of 9.0 and a temperature of 35 °C, the culture was shaken at different speeds (the speeds were set at 100 rpm, 150 rpm, and 180 rpm respectively) for 72 h. The OD of the bacterial solution obtained under different speed culture conditions was measured every 6 h. 600 value.

[0068] As shown in Table 4, Cronobacter malonaticus H4 could grow normally under the condition of a speed of 100 - 180 rpm, and it entered the logarithmic growth phase of microorganisms earliest under the culture condition of a speed of 180 rpm. Therefore, its optimal shaker speed was 180 rpm.

[0069] Table 4 OD values of the bacterial suspension of Cronobacter malonate H4 obtained at different rotation speeds 600 value

[0070]

[0071] (3) Temperature optimization for the cultivation of Cronobacter malonate H4

[0072] Cronobacter malonate H4 was inoculated into LB liquid medium at a volume ratio of 2% respectively, and cultured with shaking for 72 h under the culture conditions of pH 9.0, rotation speed of 180 rpm, and different temperatures (25 °C, 30 °C, 35 °C, and 40 °C). The OD value of the bacterial suspension obtained under different culture temperature conditions was measured every 6 h. 600 value

[0073] As shown in Table 5, the temperature tolerance range of Cronobacter malonate H4 is 25 °C - 40 °C, and it enters the logarithmic growth phase earlier at 30 °C. Therefore, relatively speaking, the suitable culture temperature is 30 °C.

[0074] Table 5 OD values of the bacterial suspension of Cronobacter malonate H4 obtained at different temperatures 600 value

[0075]

[0076] Example 3: Application of Cronobacter malonate H4 in the degradation of PTA wastewater

[0077] 1. Strain cultivation:

[0078] Cronobacter malonate H4 was inoculated into 100 mL of LB liquid medium at an inoculation amount of 2% by volume, and cultured with shaking at 30 °C, pH 9.0, and rotation speed of 180 rpm for 18 h to make the concentration of the bacterial suspension in the medium reach an OD of about 1.0. At this time, the bacterial concentration was 2.0×10 600 CFU / mL; 9 CFU / mL;

[0079] 2. Wastewater treatment:

[0080] Bacterial suspensions obtained in step 1 with different volumes (40 mL, 60 mL, 80 mL, 100 mL) were taken respectively. After centrifugation, the bacterial cell precipitate was added to 100 mL of PTA wastewater and treated under the conditions of temperature 30 °C and rotation speed 180 rpm (the treatment conditions are the optimal growth conditions of strain H4) for 72 h. The COD content of the wastewater before and after treatment was detected, and the COD degradation rate of strain H4 to PTA wastewater was calculated. The original COD value of the wastewater was 90000 - 100000 mg / L, and the pH was 8.76.

[0081] As can be seen from Table 6, when the dosage of Cronobacter malonate H4 is 80 mL, the efficiency of degrading PTA wastewater can reach the maximum, which is 76.73%.

[0082] Table 6 COD degradation rate of PTA wastewater treated with different dosages of Cronobacter malonate H4

[0083]

[0084] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered by the scope of the claims of the present invention.

Claims

1. A strain of Cronobacter malonaticus H4, characterized in that The deposit number is CGMCC No.29958.

2. Application of Cronobacter malonate H4 according to claim 1 in degrading COD in PTA wastewater.

3. Use of the malonate Cronobacter H4 described in claim 1 in the preparation of a microbial preparation for degrading COD in PTA wastewater.

4. A microbial preparation containing the malonate Cronobacter H4 according to claim 1.

5. Application of the microbial preparation according to claim 4 in degrading COD in PTA wastewater.

6. Use of the malonate Cronobacter H4 according to claim 1 or the microbial preparation according to claim 4 in the preparation of a PTA wastewater COD treatment agent.

7. A method for degrading COD in PTA wastewater, characterized in that: The method comprises the following steps: inoculating the malonate Cronobacter H4 described in claim 1 into a liquid culture medium, culturing the culture medium to a logarithmic growth phase to obtain a bacterial liquid, and adding the bacterial bodies obtained by centrifuging the bacterial liquid into PTA wastewater for treatment.

8. The method according to claim 7, characterized in that The culture is carried out in a constant temperature shaker at a pH of 7-11, a temperature of 25-40° C., and a rotation speed of 100-180 rpm.

9. The method according to claim 7, characterized in that: The treatment is carried out at a pH of 7-11, a temperature of 25-40° C., and a rotation speed of 100-180 rpm for 72 hours.

10. The method according to claim 9, characterized in that The treatment conditions are pH 9.0, temperature 30° C., and rotation speed 180 rpm.

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