Triethylamine-degrading bacterium and application thereof

Abstract

This invention discloses a triethylamine-degrading bacterium and its applications, solving the problems of low triethylamine concentration tolerance and long retention time in wastewater from existing triethylamine-degrading strains. The bacterial strain used in the biological pretreatment technology of this invention is *Sphingomyelin-Methophilus* P1, which was deposited on December 17, 2020, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.21389, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The triethylamine-degrading bacterium of this invention exhibits good concentration tolerance, initiating the reaction in wastewater with a triethylamine concentration of 2000 mg / L. Under aerobic conditions, the triethylamine degradation rate in the wastewater reaches 99% within 24 hours, effectively reducing the organic load concentration in the wastewater and achieving better treatment results.

Description

Technical Field

[0001] This invention relates to the field of biotechnology for wastewater treatment, specifically a biological treatment method for high-concentration triethylamine wastewater. Background Technology

[0002] Triethylamine (TEA) is an aliphatic amine widely used as an auxiliary raw material in chemical production. For example, it is used as a polymerization inhibitor in the production of tetrafluoroethylene, as a purification solvent for penicillin and tetracycline in antibiotic production, as a depolymerizing agent for paraformaldehyde in glyphosate production, and as a catalyst in the synthesis of carbamate pesticides. Furthermore, triethylamine is an important raw material for pesticides such as cypermethrin, imidacloprid, phosphamidon, and chlordane. It can also be used as a high-energy fuel and liquid rocket propellant. In recent years, with the rapid development of the pharmaceutical and pesticide industries, the scope and scale of triethylamine use have continuously expanded, leading to a rapid increase in the total amount emitted into the environment. In China alone, the annual discharge of triethylamine-containing wastewater from glyphosate production reaches approximately 50 million tons. During industrial production and use, triethylamine can irritate the skin and mucous membranes, and also affect the liver, kidneys, and cardiovascular system. Triethylamine entering the environment causes serious environmental pollution and has various toxic effects on humans and animals. It inhibits the synthesis of macromolecules such as DNA, RNA, and proteins, and also has teratogenic effects on animal embryonic cells. With rapid industrial development, the environmental problems caused by the widespread use of triethylamine are becoming increasingly serious.

[0003] Currently, the main effective treatment processes for triethylamine-containing wastewater in production are physicochemical methods, such as ozone oxidation and activated carbon adsorption used by Shen Jinzhong et al. However, physicochemical treatment methods are generally costly. In contrast, biological treatment has the advantages of low cost, high efficiency, and no secondary pollution. The Environmental Data Sheet for Organic Compounds indicates that even with acclimated sludge, the degradation performance of triethylamine is not very good. Wang et al., using acclimated sludge, could only completely degrade triethylamine concentrations below 200 mg / L, requiring 114.4 hours. The reported triethylamine-degrading strains, *Pseudomonas citronellolis* and *Mycobacterium dien-hoferi*, also have low degradation efficiencies, requiring 4 days to completely degrade 50 mg / L of triethylamine. Summary of the Invention

[0004] The purpose of this invention is to provide a triethylamine-degrading bacterium and its application to solve the problems of low triethylamine concentration tolerance and long retention time in existing triethylamine-degrading strains. The triethylamine-degrading bacterium screened in this patent has a better concentration tolerance ability and can initiate the reaction in wastewater with a triethylamine concentration of 2000 mg / L. Under aerobic conditions, the triethylamine degradation rate in the wastewater reaches 99% in 24 hours, effectively reducing the organic load concentration in the wastewater and achieving a better treatment effect.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A triethylamine-degrading bacterium, characterized in that: the triethylamine-degrading bacterium is *Sphingobacterium thalpophilum* P1, deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 17, 2020, with accession number CGMCC NO.21389, and deposited at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The present invention also provides the application of the above-mentioned triethylamine degrading bacteria in fine chemical wastewater containing triethylamine.

[0008] Preferably, the above application involves the targeted addition of triethylamine-degrading bacteria to a wastewater system subjected to triethylamine wastewater impact, wherein the concentration of the added triethylamine-degrading bacteria suspension is 10. 8 MPN / ml-10 10 The dosage is MPN / m, and the addition method is twice a week, with each addition being 0.1-0.3‰V / V of the treated water volume, for a continuous period of one month.

[0009] Preferably, the concentration of the bacterial strain in the triethylamine-degrading bacteria suspension used in the fine chemical wastewater process is 10. 8 MPN / ml-10 10 MPN / ml; the dosage is twice a week, with each addition being 0.1-0.3‰V / V of the treated water volume, for a continuous period of one month.

[0010] Preferably, triethylamine-degrading bacteria are used in the aerobic stage, with dissolved oxygen at 4-6.

[0011] Preferably, the concentration of triethylamine in the fine chemical wastewater is 200-2000 mg / L.

[0012] Microbial preservation for patented procedures:

[0013] Date of deposit: December 17, 2020;

[0014] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections;

[0015] Accession number: CGMCC NO.21389;

[0016] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0017] Classification and nomenclature (strain) of Institute of Microbiology, Chinese Academy of Sciences: Sphingobacterium thalpophilum P1.

[0018] Compared with the prior art, the present invention provides a biological treatment method for high-concentration triethylamine wastewater, achieving the following technical effects: 1) The triethylamine-degrading bacterial strain *Sphingobacterium thalpophilum* P1 can use triethylamine as the sole carbon and nitrogen source and mineralize it into ammonia nitrogen; 2) The triethylamine-degrading bacterial strain *Sphingobacterium thalpophilum* P1 can degrade high-concentration triethylamine. For example, it can initiate the reaction in wastewater with a triethylamine concentration of 2000 mg / L. Under aerobic conditions, the triethylamine degradation rate in the wastewater reaches 99% in 24 hours, which is far higher than the 100 mg / L of the prior art. Attached Figure Description

[0019] Figure 1 A comparative diagram showing the anaerobic triethylamine degradation activities of two bacteria, *Triethylamine-degrading bacteria* and *Water Country Denitrifying Bacteria*.

[0020] Figure 2 A comparative diagram of the aerobic triethylamine decomposition activities of two bacteria, triethylamine-degrading bacteria and water-land denitrifying bacteria. Detailed Implementation

[0021] The present invention will be described below through specific embodiments, but the present invention is not limited thereto. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and materials described are commercially available unless otherwise specified. The following embodiments are not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of protection of this patent.

[0022] Example 1

[0023] Isolation of strains

[0024] The isolation of the strain can be referenced in patent CN201811383781.1, the only differences being: the source is triethylamine wastewater; and the culture medium used is different.

[0025] The culture medium used during the acclimatization process was as follows: 0.2g MgSO4, 0.02g KCl, 1g KH2PO4, 2.6g K2HPO4, 1000mL distilled water, 40g washed agar, pH 7.5-8. 0.3g triethylamine was added after sterilization and cooling to 40℃.

[0026] Identification of strain morphology and physiological and biochemical levels

[0027] When the strain of this invention grows on solid LB medium, the colonies are round, pale yellow, opaque, and wrinkled; the bacterial cells are Gram-negative and short rod-shaped. This strain can grow using triethylamine as the sole carbon and nitrogen source, and the initial concentration can be adjusted.

[0028] Formaldehyde at a concentration of 400 mg / L was completely degraded within 24 hours.

[0029] Identification at the molecular biological level:

[0030] Bacterial DNA was extracted using a DNA extraction kit (Sangon) following standard extraction procedures, followed by PCR amplification of the target fragment. A pair of universal primers was designed for amplifying the 16S rDNA sequence:

[0031] 27F 5'-AGAGTTTGATCMTGGCTCAG-3';

[0032] 1492R 5'-GGTTACCTTGTTACGACTT-3';

[0033] Using genomic DNA as a template, Premix Tap™ was added for PCR amplification. The PCR product was detected by 1% agarose gel electrophoresis, purified using a DNA purification and recovery kit, ligated into the pGM-T vector, transformed into E. coli DH5α competent cells, plated onto LB solid medium containing ampicillin, and cultured at 37°C for 12 h. Colonies were picked and cultured in liquid LB medium, shaken at 37°C and 180 rpm overnight. Plasmids were extracted using a plasmid extraction kit and analyzed.

[0034] The PCR reaction conditions were as follows: 94℃ pre-denaturation for 5 min; followed by 30 cycles of 94℃ denaturation for 1 min; annealing at 55℃ for 1 min; and extension at 72℃ for 5 min. The PCR amplification products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequence is shown in SEQ ID NO.1. The strain was identified by 16S rDNA, and the results were compared for homology using the Genebank Blast program. The results showed that the strain shared 99% homology with *Sphingobacterium thalpophilum*, further confirming that this strain is *Sphingobacterium thalpophilum*. This strain was deposited on December 17, 2020, at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC NO.21389.

[0035] Example 2

[0036] Triethylamine-degrading bacteria were inoculated into triethylamine wastewater, and the degradation of triethylamine and the transformation patterns of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen were observed.

[0037] Experimental data on the degradation of triethylamine wastewater by triethylamine-degrading bacteria

[0038]

[0039]

[0040]

[0041] Note:

[0042] 1. Simulated wastewater formula: 1g NaCl, 0.2g KH2PO4, 0.3g K2HPO4, 0.05g CaCl2, 0.02g MgSO4·7H2O, 1000ml tap water. Add triethylamine according to the influent triethylamine concentration load and adjust pH to 7.5.

[0043] 2. Anaerobic degradation of triethylamine experiment: The experiment was conducted by attaching the bacterial strain to activated carbon packing. A 1L beaker was used, and 6-8 mesh granular activated carbon was added to the 250ml mark. 10ml of a 10% concentration was then added. 8 A CFU / m solution of triethylamine-degrading bacteria was added to 1L of simulated wastewater. One reaction cycle was 24h, and the ammonia nitrogen value and triethylamine concentration were measured every 24h.

[0044] 3. Aerobic Degradation of Triethylamine Experiment: The bacterial strain was mounted on polyurethane packing material (RAS-specific biological packing material from Shandong Banghao Environmental Protection Technology Co., Ltd., with a shape and specifications of 22±1mm and an expansion coefficient of 1.2 after water absorption) in a biological fluidized bed configuration. A 1L graduated cylinder was used, with an aeration head installed at the bottom and fixed with ceramic balls. The polyurethane packing material, after absorbing water and expanding, was added to the 250ml mark, followed by 10ml of a 10% concentration solution. 8 A CFU / m solution of triethylamine-degrading bacteria was added to 1L of simulated wastewater, and aeration was carried out at the bottom. The pH was controlled at 6-9 and the dissolved oxygen at 4-8. One reaction cycle was 24h, and the ammonia nitrogen value and triethylamine concentration were measured every 24h.

[0045] summary:

[0046] 1. The triethylamine-degrading bacteria obtained through domestication and screening enhanced both the anaerobic and aerobic stages when degrading simulated water with triethylamine as the single pollutant.

[0047] 2. During the anaerobic degradation of triethylamine by triethylamine-degrading bacteria, a portion of triethylamine cannot be completely decomposed, resulting in a significant accumulation of organic amine intermediates.

[0048] 3. When the initial triethylamine concentration is less than 2000 mg / L, the triethylamine-degrading bacteria aerobically degrade triethylamine quite thoroughly, with almost no accumulation of organic amine intermediates.

[0049] 4. In addition, the applicant's preliminary study on the triethylamine degradation characteristics of triethylamine-degrading bacteria revealed that the activity of the bacteria in degrading triethylamine first increased and then decreased with the increase of the initial triethylamine concentration. When the triethylamine concentration reached 300 mg / L, the degradation rate of the strain reached its highest value. Specifically, for aerobic degradation, when the triethylamine concentration was ≤300 mg / L, the degradation rate reached 100%. When 300 mg / L < triethylamine concentration ≤2000 mg / L, the degradation rate only decreased slightly and remained above 99%. When the influent pH was ≤5 or ≥10, the triethylamine-degrading bacteria showed almost no degradation activity. When the pH was between 6 and 9, the degradation of triethylamine by the bacteria was better, and the degradation activity was highest when the influent pH was 8.

[0050] Example 3

[0051] To further evaluate the treatment effect of triethylamine-degrading bacteria on triethylamine wastewater, this experiment conducted a small-scale comparative experiment on triethylamine-degrading bacteria and Shuizhiguo denitrifying bacteria agent (purchased from Wuhan Shuizhiguo BioPower110PLUS compound denitrifying bacteria), and analyzed the experimental results.

[0052] Triethylamine-simulated wastewater was subjected to degradation experiments using the A / O process, and the results were compared with existing denitrifying bacteria in the laboratory.

[0053] Preparation of simulated water: 1g NaCl, 0.1g KH2PO4, 1000ml tap water, add triethylamine according to the triethylamine concentration load of the influent, pH=7.5.

[0054] The A-stage process involved loading the microbial strain onto activated carbon packing material. Three 1L Erlenmeyer flasks, labeled A1, A2, and A3, were used. 250ml of 6-8 mesh granular activated carbon was added to each flask. Additionally, 10ml of 10% activated carbon was added to each of the flasks. 8 Triethylamine (CFU / m³) and seed culture of *Aqua davidii* (a type of denitrifying bacteria) were added to simulated water. One reaction cycle was 48 hours, with ammonia nitrogen and triethylamine concentrations measured every 24 hours. The O-stage process involved attaching the bacterial strain to polyurethane packing material and conducting an oxidation experiment. Three 1L graduated cylinders were used, labeled O1, O2, and O3. Aeration heads were placed at the bottom of the cylinders and fixed with ceramic balls (to cut air bubbles and evenly disperse them in the simulated water). The polyurethane packing material, after absorbing water and expanding, was added to the 250ml mark. 10ml of a 10% concentration was added to each of O1, O2, and O3. 8 Triethylamine (CFU / m³), seed culture of *Narachidonia denitrifying bacteria*, and simulated water were added. One reaction cycle was 24 hours, and the concentrations of ammonia nitrogen and triethylamine were measured every 24 hours.

[0055] Depend on Figure 1 , Figure 2 (TEA in the figure refers to triethylamine degrading bacteria) It can be seen that the apparent decomposition rate of triethylamine in the anaerobic and aerobic stages of TEA is higher than that of the denitrifying bacteria in the water country, indicating that the triethylamine degrading bacteria obtained through domestication, screening and compounding have enhanced both the anaerobic and aerobic stages when degrading simulated water with triethylamine as the single pollutant.

Claims

1. A triethylamine-degrading bacterium, characterized in that: The triethylamine-degrading bacterium is *Sphingomonas ferruginosa* (…). sphingobacterium thalpophilum P1, deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 17, 2020, with accession number CGMCC NO.21389, and located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The application of the triethylamine-degrading bacteria as described in claim 1 in fine chemical wastewater containing triethylamine, characterized in that: The application is to degrade triethylamine in fine chemical wastewater.

3. The application of the triethylamine-degrading bacteria as described in claim 2 in fine chemical wastewater containing triethylamine, characterized in that: The triethylamine degradation bacteria is added to the wastewater system impacted by triethylamine wastewater, the concentration of the strain in the bacterial suspension is 10 8 MPN / ml-10 10 MPN / m, the adding mode is twice a week, the adding amount is 0.1-0.3‰V / V of the treated water, and the process is continuous for one month.

4. The method according to claim 2, characterized in that, The concentration of triethylamine in the fine chemical wastewater is 200-2000 mg / L.

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