Pseudomonas veronii for degrading n -methyl-2-pyrrolidone

Abstract

The invention relates to the bacterial strain Pseudomonas veronii T610 with accession number NCAIM P (B) 001522, which is capable of degrading high concentrations of N-methyl-2-pyr- rolidone (NMP), thereby removing NMP from the environment, cleaning a medium, such as an aqueous medium, contaminated with NMP, and bioremediation. The invention also relates to the use of the bacterial strain Pseudomonas veronii T610 with accession number NCAIM P (B) 001522 for degrading NMP, especially in wastewater containing NMP.

Description

[0001] PSEUDOMONAS VERONII FOR DEGRADING N -METHYL-2-PYRROLIDONE

[0002] FIELD OF THE INVENTION

[0003] The invention relates to the bacterial strain Pseudomonas veronii T610 with accession number NCAIM P (B) 001522, which is capable of degrading high concentrations of N-methyl-2-pyr- rolidone (NMP), thereby removing NMP from the environment, cleaning a medium, such as an aqueous medium, contaminated with NMP, and bioremediation. The invention also relates to the use of the bacterial strain Pseudomonas veronii T610 with accession number NCAIM P (B) 001522 for degrading NMP, especially in wastewater containing NMP.

[0004] BACKGROUND OF THE INVENTION

[0005] With the development of the electric vehicle industry, the demand for lithium-ion batteries is increasing continuously. The professional, efficient and environment-friendly treatment of industrial wastewater generated during production is of paramount importance. During the production of batteries, toluene, carbonates (diethyl carbonate (DEC), ethylene carbonate (EC), dimethyl carbonate (DMC), ethyl methyl carbonate (EMC)) and N-methyl-2-pyrrolidone (NMP) are used as solvents. Further, industrial wastewater produced during battery production has a high lithium and salt content.

[0006] NMP belongs to the group of aprotic solvents, it is a yellowish liquid, that is miscible with water. Its low volatility and flammability contribute to its wide range of uses (e.g. : battery production, paint industry, polymer production, pharmaceutical industry) (Jouyban, A., et al. (2010). Review of Pharmaceutical Applications of A-Methyl-2-Pyrrolidone. Journal of Pharmacy & Pharmaceutical Sciences, 13(4), 524.). NMP has been shown to be harmful to human health, primarily due to its effects on fertility and the fetus, and its use is being regulated in both Europe and the United States. According to the literature, NMP can be degraded by physicochemical methods, but several factors affect their efficiency. Existing physicochemical technologies are expensive and not commercially attractive. In addition, these processes only transfer contaminants from one phase to another, rather than removing them (Muruganandham, M., et al. (2007). Mineralization of A-methyl-2-pyrolidone by advanced oxidation processes. Separation and Purification Technology, 55(3), 360-367.).

[0007] Biological wastewater treatment is an efficient and environment-friendly technology, which is fast and cost-effective for the pollutants mentioned above, especially NMP. However, this requires a microorganism capable of inactivating, removing NMP and treating the polluted medium. BRIEF DESCRIPTION OF THE INVENTION

[0008] The invention provides bacteria belonging to the T610 strain of Pseudomonas veronii, deposited with the accession number NCAIM P (B) 001522.

[0009] The invention further relates to bacteria capable of degrading N-methyl-2-pyrrolidone, the bacteria generated from bacteria belonging to the T610 strain of Pseudomonas veronii by culturing or genetic modification.

[0010] The use of bacteria belonging to the T610 strain of Pseudomonas veronii, having the accession number NCAIM P (B) 001522, for degrading N-methyl-2-pyrrolidone is also provided. Preferably the A-methyl-2-pyrrolidone is in an aqueous medium.

[0011] Preferably the aqueous medium is wastewater, preferably surface or subsurface water.

[0012] Preferably A-methyl-2-pyrrolidone has been introduced into the aqueous medium during the production of a battery.

[0013] BRIEF DESCRIPTION OF THE FIGURES

[0014] Figure 1 Degradation of NMP by Pseudomonas veronii T610 strain in nitrogen-free medium Figure 2 Pseudomonas veronii T610 strain 1 / 2 TSA plate

[0015] Figure 3: Optical density values of the T610 strain incubated at 6-37 °C as measured at 600 nm Figure 4: Results of the pathogenicity testing of strain T610

[0016] Figure 5: 16S rDNA sequence (SEQ ID No 1) of isolate T610

[0017] DETAILED DESCRIPTION OF THE INVENTION

[0018] The investigation of several Hungarian surface and groundwaters contaminated with volatile organic compounds (VOCs) led to the isolation of the NMP-degrading bacterial strain Pseudomonas veronii (T610). The colony formed by the isolated bacteria showed a significant increase in optical density (measured at 600 nm) in a nitrogen-free minimal nutrient solution containing 500 mg / l NMP (Figure 1).

[0019] The strain was deposited in the National Collection of Agricultural and Industrial Microorganisms (1118 Budapest, Somloi út 14-16., Hungary) withthe accession number NCAIM P (B) 001522 on June 13, 2024.

[0020] The T610 strain is Gram -negative and forms beige coloured, round and convex colonies with jagged edges on ½ TSA plates (Figure 2).

[0021] The T610 strain is capable of degrading NMP.

[0022] Upon inoculating the T610 strain into a medium containing NMP, ammonium and / or methylamine are produced from NMP, which are degraded in the sample after further incubation. The daughter compound, from which methylamine is separated, enters the Krebs-Szent-Gyorgyi cycle, during which carbon dioxide, water and biomass are produced. The degradation process is extremely rapid, without the accumulation of toxic compounds. The T610 strain is capable of complete degradation (mineralization) of NMP under denitrifying, anoxic conditions. During the experiments aimed at investigating this, no significant accumulation of NMP daughter compounds or nitrite was observed.

[0023] Strain T610 was able to degrade NMP at 30 g / l under aerobic conditions. To the best of our knowledge, only three bacterial strains (Pseudomonas sp. MS2 (Zhang, Q., et al. (2016). A controlled wet-spinning and dip-coating process for preparation of high -permeable TiO2hollow fiber membranes. Water Science and Technology, 73(4), 725-733.), Alicycliphilus sp. BQ1, Alicycliphilus sp. BQ8 (Oceguera-Cervantes, A., et al. (2007). Characterization of the Polyu- rethanolytic Activity of Two Alicycliphilus sp. Strains Able To Degrade Polyurethane and N- Methylpyrrolidone. Applied and Environmental Microbiology, 73(19), 6214-6223.) have been reported to date to be able to tolerate and also degrade a maximum of 25 g / l NMP. It can be stated that strain T610 is the microorganism that is capable of degrading NMP at the highest concentration.

[0024] The thermal optimum of the T610 strain is at 25 °C; the strain showed the most intensive OD600growth at this temperature. T610 has shown growth in the temperature range of 6-30 °C, we did not experience an increase in the OD600value in any of the samples at 37 °C, which is a very advantageous feature of the bacteria, since it is therefore not expected to be a human pathogen. Since the strain showed activity even at 6 °C, it can be considered psychrotolerant. The wide temperature tolerance of the strain is ideal for both industrial and possible field remediation.

[0025] The T610 strain is able to grow in a very wide pH range (pH=3.5-10.0), which allows for a wide range of industrial applications.

[0026] The T610 strain is able to grow at a salt concentration of 60 g / l and is moderately halotol erant. Due to its salt tolerance, the T610 strain can be used not only for the biological treatment of NMP-containing wastewater generated during the production of electrode layers and tank washing, but also for the biological treatment of industrial wastewater produced during the discharge of waste cells in battery production, which contain NMP and carbonates further to a salt concentration of 60 g / l.

[0027] The main data of Pseudomonas veronii T610 are summarized in Table 1.

[0028] 1. tablazat

[0029] Example

[0030] Isolation of the T610 strain

[0031] To isolate the NMP-degrading bacteria, we used several volatile organic compounds (VOCs) containing surface and groundwater samples from Hungary, in which we intended to propagate and then isolate the NMP-degrading microorganisms during an enrichment procedure.

[0032] There was only one culture (from an enrichment prepared from a groundwater sample from the Central Hungary region) showing growth at the end of the enrichment procedure, from which we succeeded in isolating a unique colony that was able to show a significant increase in optical density (measured at 600 nm) in a nitrogen-free minimal medium containing 500 mg / l NMP. The results of the triplicate batch microcosm experiment were plotted on a diagram, the individual points were formed from the average of the measured values, the error bars indicate the standard deviation of the triplicate samples (Figure 1). The isolated strain, which was named T610, is able to grow on NMP as the sole carbon, nitrogen and energy source., from which we succeeded in isolating a unique colony that was able to show a significant increase in optical density (measured at 600 nm) in a nitrogen-free minimal medium containing 500 mg / l NMP. The results of the triplicate batch microcosm experiment were plotted on a diagram, the individual points were formed from the average of the measured values, the error bars indicate the standard deviation of the triplicate samples (Figure 1). The isolated strain, which was named T610, is able to grow on NMP as the sole carbon, nitrogen and energy source.

[0033] For the 16S rDNA-based identification of the T610 isolate, the 16S rDNA of an individual colony of the T610 strain was amplified using colony PCR, and the 16S rDNA sample amplified in the PCR device was determined (SEQ ID NO 1).

[0034] Culture media used in the experiments

[0035] The culture plates and culture media prepared during the studies were sterilized in an autoclave for 15 minutes at 121 °C. The nitrogen-free MSM culture medium and culture plate contain components that react with each other to form precipitates, so a separate solution was prepared from these components, taking into account their solubility in water.

[0036] Nitrogen-free MSM culture medium and culture plate

[0037] The composition of the nitrogen-free MSM is described in Table 2.

[0038] Table 2

[0039] MSM nitrogen-free medium

[0040] The components of the nitrogen-free MSM microelement solution are described in Table 3.

[0041] Table 3

[0042] MSM microelement solution

[0043] The described microelement solution was sterilized with a 0.22 μm pore size syringe filter, then added to the cooled nutrient solution in a final volume of 1 ml. NMP was pipetted into the nutrient solution and nutrient plate prepared for the study of NMP-degrading microorganisms at the desired concentration before sterilization.

[0044] ½ Tryptone-soy culture plate

[0045] The composition of the ½ TSA culture plate is described in Table 4.

[0046] Table 4

[0047] ½ TSA culture plate

[0048] Determination of NMP and its degradation products by HPLC measurement

[0049] NMP-containing samples were tested by routine HPLC. The NMP content of the samples was determined by a HPLC-DAD measurement. The sample was stored at -20 °C until measurement. After warming to room temperature, the sample was centrifuged at 13,000 rpm for 10 minutes, then the supernatant was filtered through a 0.22 μm CA syringe filter, diluted with eluent if necessary and measured by the HPLC-DAD method. An Agilent Technologies 1260 Infinity high-performance liquid chromatograph coupled with an Agilent DAD detector was used for the measurements. The automatic sample injector (Agilent Technologies 1260 ALS) injected 10 μl of the samples. The measurement was performed isocratically at a flow rate of 0.7 ml / min. A mixture of acetonitrile and distilled water in a ratio of 5 :95 was used as the eluent. The separation was performed on a Zorbax Eclipse Plus C18 (250 x 4.6 mm, 5 μm) column, the temperature of which was 25 °C. The detection was performed at 200 nm with a reference wavelength of 550 nm. The analysis time was 15 min, the measuring range of the method was 0.25-500 mg / l, and the measurement accuracy was ±10%.

[0050] Determination of anion (nitrate and nitrite) concentration of liquid samples

[0051] The samples were stored at -20 °C until the start of the analysis. During sample preparation, the samples were centrifuged at 13,000 rpm for 10 minutes, then diluted appropriately from the supernatant to a final volume of 1 ml in vials (Agilent Technologies, AMBER, 2 ml, 9 mm S / T) by adding fluoride internal standard. After preparation, the vials were placed in the sample holder of the instrument.

[0052] The automatic sampler (Shimadzu SIL-10AF) injected 20 pl of the samples. The measurement was performed isocratically, eluent: 1.8 mM Na2CO3 / 1.7 mM NaHCO3 buffer solution, flow rate: 1.0 ml / min, column temperature: 32 °C, the sensitivity of the conductometer was set to 100 μS. The separation was performed with a ShodexTM IC SI-90 4E (4.0 x 250 mm, 9 μm) column, an IC SI-90G (PEEK) precolumn and a SeQuant SAMS anion IC suppressor. 0.04 N H2SO4 was used as the suppressor regeneration solution. The detector was a Sykam S3111 CD. The concentrations of the following anions were examined during the measurement: nitrate and nitrite. The measuring range of the method is 1-200 mg / l, and the measurement accuracy is ±10%.

[0053] Liquid TOC / TN (TOC: Total Organic Carbon, TN: Total Nitrogen)

[0054] Samples were stored in 50 ml centrifuge tubes at +4 °C until the start of the measurement. The samples were centrifuged at 6,000 rpm for 10 minutes before measurement.

[0055] After the inorganic carbon content of the sample introduced into the device (Teledyne Tekmar Apollo 9000 TOC Combustion Analyzer + Total Nitrogen) is extracted with phosphoric acid solution (21%), it enters a combustion tube filled with a catalyst, where at 730 °C in a pure oxygen (purity: 5.0) atmosphere, the carbon content of the sample is oxidized to carbon dioxide and the nitrogen content to nitrogen monoxide during a thermocatalytic reaction.

[0056] Carbon dioxide in the gas stream is measured by an NDIR detector based on its infrared absorbance, while nitrogen dioxide, which is formed after the excitation of nitrogen monoxide with ozone, is measured by a chemiluminescent detector (CLD). The measured amount of CO2 and NO2 is proportional to the carbon and nitrogen content of the sample. The measurement range of the method is 10-1000 ppm C for TOC, and 5-100 ppm N for TN. The measurement accuracy is ±10%.

[0057] Degradation of NMP by Pseudomonas veronii T610 strain

[0058] The concentration of NMP and its degradation products was measured by high-performance liquid chromatography (HPLC). In addition to NMP, the following components were examined: 2-pyrrolidone, 5-hydroxy-N-methylpyrrolidone, N-methylsuccinimide and succinimide. The determination of ammonium nitrogen was carried out using a spectrophotometer according to the MSZ ISO 7150-1 : 1992 standard. The disadvantage of the measurement method is that it cannot separate ammonium and methylamine well, so methylamine also gives a signal, so it cannot be determined whether ammonium and / or methylamine appeared in the sample. The method gave a positive signal in several cases in NMP-containing test systems inoculated with strain T610, thus it can be stated that during the breakdown of NMP by strain T610, ammonium and / or methylamine are produced, which decompose in the sample after further incubation.

[0059] We were able to detect only methylamine and / or the ammonium formed from it as intermediate products, which are degraded upon further treatment. The daughter compound from which methylamine is separated enters the Krebs- Szentgybrgyi cycle, producing carbon dioxide, water and biomass. Although we were unable to map the degradation pathway due to the absence of intermediates, this also means that the degradation process occurs extremely quickly, without the accumulation of toxic compounds.

[0060] Anaerobic degradation

[0061] We investigated whether Pseudomonas veronii T610 is capable of degrading NMP under anoxic, denitrifying conditions. The three parallel batch microcosm systems were set up in an anaerobic box. Before use, minimal nutrient solution was bubbled with pure nitrogen for 20 minutes in an anaerobic box (gas composition: 5.0% (n / n) H2, 5.0% (n / n) CO2, ad 100% N2). Sterile-filtered NMP (500 mg / l) and nitrate (1200 mg / l nitrate in the form of potassium nitrate) were then added to the medium as an electron acceptor. To monitor possible abiotic evaporation or degradation, we prepared abiotic control samples in triplicate, which were prepared in the same way as the biotic samples, with the only difference that the abiotic samples did not contain strain T610, and these microcosms contained mercury sulfate (240 mg / l). The microcosms were incubated at 25 °C, without shaking. The microcosm systems were analyzed for NMP and degradation products, optical density (OD600) measured at 600 nm by daily measurements, anion (nitrate and nitrite) content was measured every two days, and TOC was measured weekly, and daily after the 20th day.

[0062] NMP concentration decreased from 500 mg / l to 349 mg / l by day 3 in the microcosm systems inoculated with strain T610, a trend that was continuous, so that by day 16, NMP in all samples containing strain T610 decreased below the detection limit (0.25 mg / l). The TOC content of the sample in the initial sample was 302.0 mg / l, which by day 20 averaged 44.6 mg / l in the three microcosms containing T610. By day 23, the TOC concentration of all samples inoculated with T610 decreased below the detection limit (10 mg / l). Significant accumulation of NMP daughter compounds or nitrite was not experienced in any of our experimental systems during the entire duration of the experiment. Batch microcosms started at an OD600of 0.010, which reached an average of 0.040 by day 3 and 0.100 by day 16. The highest OD600values (average 0.134) were measured on day 23.

[0063] Since the initial nitrate concentration (1200 mg / l) in the samples containing T610 decreased to below 80.50 mg / l on day 14, 500 mg / l nitrate was added to these microcosms on day 15. At the end of the experiment, an average of 135.7 mg / l nitrate remained in the systems containing T610. The highest nitrite concentrations were measured on day 8 (average 64.93 mg / l), which decreased below the detection limit after day 14.

[0064] During the entire duration of the experiment, the NMP and nitrate concentrations in the abiotic samples varied within the analytical error limit, the formation of intermediates was not detected, and the optical density of these samples did not change either.

[0065] The decrease of NMP and TOC below the detection limit, the increase of the OD600value, the decrease of nitrate, and the appearance of nitrite and its subsequent decrease below the detection limit are clear evidences that the T610 strain is capable of complete degradation (mineralization) of NMP under denitrifying, anoxic conditions.

[0066] To the best of our knowledge, strain T610 is the second bacterium, after Paracoccus pantot- rophus NJUST38, that is capable of degrading NMP under denitrifying conditions.

[0067] Investigation of the NMP tolerance of Pseudomonas veronii strain T610

[0068] The outstanding NMP-degrading potential and tolerance of the T610 strain is demonstrated by the fact that we had to modify the aerobic microcosm setup we used as per the protocol, as the T610 strain is capable of degrading such a large amount of NMP that the 5.2-fold air space above the liquid we routinely use was no longer sufficient. Thus, in batch systems, for concentrations greater than 1 g / l, we ensured the oxygen supply during incubation with atomizer stones and air pumps.

[0069] The NMP tolerance of strain T610 was tested in nitrogen-free MSM medium. In order to provide the necessary oxygen supply in the microcosms, we used compressed air passed through a sterile filter, which was introduced into atomizing stones and, in order to achieve a higher dissolution efficiency, the systems were mixed with magnetic stirrers. The air introduced by the pumps was removed through a side-outlet syringe needle pierced through the Teflon -lined septum after the systems were started. The microcosms were incubated under a sterile hood at 25 °C. To monitor possible abiotic evaporation or degradation, abiotic control samples were prepared in triplicate, which contained nitrogen-free MSM, 30 g / l NMP, and mercuric sulfate (240 mg / l). The concentration of NMP was monitored by HPLC after 8 weeks of incubation, at the the same time as the OD600measurement was performed.

[0070] After the start of the experiment, a visible increase occurred after 10 days; OD600subsequently increased continuously, although more slowly than usual. The 30 g / l NMP concentration clearly inhibited the growth of T610, but after a few days the strain was able to adapt to the high NMP concentration and then start to break down NMP. After eight-week incubation, the OD600value was 0.521, and the NMP concentration decreased below the detection limit. In the abiotic sample, the NMP concentration varied within the analytical error limit, and no difference in optical density was observed.

[0071] Strain T610 was able to degrade NMP at 30 g / l under aerobic conditions, which is the highest tolerance level of the strain to our current knowledge. To the best of our knowledge, only three bacterial strains (Pseudomonas sp. MS2, Alicycliphilus sp. BQ1, Alicycliphilus sp. BQ8) have been described to date as being able to tolerate and degrade 25 g / l NMP. No microorganism or consortium was found in the literature that was described as being able to tolerate and degrade NMP concentrations higher than 25 g / l, so it can be stated that the T610 strain is the one that is able to degrade NMP at the highest concentration.

[0072] Determination of the thermal optimum of Pseudomonas veronii T610 strain

[0073] The temperature tolerance and optimum of the isolated bacteria was investigated in triplicate samples in 500 mg / l NMP, nitrogen-free medium, at temperature points of 6 °C, 10 °C, 15 °C, 25 °C, 30 °C and 37 °C. During the experiment, samples were taken from the systems every six hours for 60 hours. The growth of strain T610 was monitored by the change in optical density (OD600) measured at 600 nm, the results are presented in Figure 3.

[0074] Pathogenicity of Pseudomonas veronii T610 strain

[0075] In terms of human pathogenicity, Pseudomonas veronii species can be classified as risk group 1 accessed 02 szept- ember 2024) which is also supported by the fact that the strain does not grow at 37 °C. Furthermore, we examined the pathogenicity of the strain in the PathogenFinder (https: / / cge.food.dtu.dk / services / PathogenFinder / ) program. The program estimates the probability of a given bacterium being a human pathogen based on the 16S rDNA. The results of the study (Figure 4) coincide with the previous ones, so it can be safely stated that the T610 strain is not a human pathogen. pH tolerance of the T610 isolate

[0076] The the pH optimum and tolerance of Pseudomonas veronii T610 was examined in the range of 3.0 and 11.0, in 0.5 pH steps. The pH tolerance (pH=3.5-10.0) and pH optimum (pH=5.0- 7.5) of the T610 strain were determined by monitoring the change in optical density (OD600) of the samples measured at 600 nm.

[0077] Salt tolerance of the T610 isolate

[0078] The salt tolerance of Pseudomonas veronii T610 was determined in the range of 0-100 g / l, in 10 g / l steps. By monitoring the change in optical density (OD600) of the samples measured at 600 nm, it can be determined that the T610 strain is able to grow at a salt concentration of 60 g / l. NMP-degrading potential of related strains

[0079] We examined the NMP-degrading capacity (500 mg / l NMP) of Pseudomonas aeruginosa 785, Pseudomonas fluorescens. Pseudomonas sp. BX1, Pseudomonas sp. EMI and Pseudomonas sp. LH1G9 (proposed name: Pseudomonas veronii LH1G9) strains under aerobic and facultative anaerobic conditions. The preparation and incubation of the systems were the same as the routine used for the T610 strain.

[0080] The study was continued for two weeks, during which the optical density (OD600) of the samples measured at 600 nm and the change in NMP concentration were monitored. We did not experience an increase in the OD600value, nor a decrease in the NMP concentration within the analytical error limit in any of the samples, thus confirming that none of the tested strains is capable of biodegrading NMP.

Claims

CLAIMS1. Bacteria belonging to Pseudomonas veronii T610 strain, having the accession number NCAIM P (B) 001522.

2. Bacteria produced by culturing or genetically modifying the bacteria according to claim 1 and capable of degrading A-methyl-2-pyrrolidone.

3. Use of the bacteria according to claim 1 or 2 for degrading A-methyl-2-pyrrolidone.

4. The use according to claim 3, wherein A-methyl-2-pyrrolidone is in an aqueous medium.

5. The use according to claim 4, wherein the aqueous medium is wastewater, preferably surface or subsurface water.

6. The use according to any one of claims 3-5, wherein N-methyl -2 -pyrrolidone has been introduced into the aqueous medium during the production of a battery.

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