Acinetobacter venetianus PFZR-1 and its application in the degradation of organic pollutants

Abstract

The present invention discloses an Acinetobacter venetianus PFZR-1 and its application in the degradation of organic pollutants. The Acinetobacter venetianus PFZR-1 of the present invention can grow using cyclohexane as the sole carbon source and energy source, and can degrade cyclohexane at a concentration of up to 316.4 mg / L. Moreover, the suitable growth pH range of this bacterium is relatively wide, and it can degrade cyclohexane well at pH values of 6.0 to 8.0, with a degradation rate of over 90%. In addition, the Acinetobacter venetianus PFZR-1 has a high degradation efficiency for cyclohexane, low cost, simple operation, and does not produce secondary pollution, having more advantages than traditional adsorption and photocatalytic degradation. It provides strong technical support for the microbial purification engineering applications of soil, waste gas, and wastewater containing this pollutant.

Description

(1) Technical Field

[0001] The present invention relates to an Acinetobacter venetianus PFZR-1 and its application in the degradation of organic pollutants. (2) Background Art

[0002] Hydrocarbons are organic compounds composed of only carbon and hydrogen elements. They are all insoluble in water, soluble in organic solvents, and have a density smaller than that of water. Therefore, they appear as oily insoluble substances in water. Cyclohexane (C6H6) is a colorless liquid with a pungent odor and is a volatile cycloalkane. Cyclohexane has a wide range of uses. It can be used as a raw material for the production of products such as cyclohexanol, cyclohexanone, and adipic acid. It can be used as a solvent for chromatographic analysis and organic synthesis processes. It can be used as an adhesive in the production of resins, coatings, etc. Therefore, cyclohexane will be widely present in the environment during related industrial production, analytical applications, transportation leaks, etc. Cyclohexane belongs to low-toxic substances, but it has a mild irritating effect on the eyes and respiratory tract. Skin contact can cause itching, and inhalation poisoning may cause anesthetic symptoms such as dizziness, nausea, and anxiety.

[0003] Cyclohexane shows a certain viscosity, so it is more likely to adhere to adsorbents in environments such as soil, surface water, and the ocean, and is often more difficult to remove. Microorganisms are also widely present in the environment. Microbial treatment of pollutants has received extensive attention due to its advantages such as low cost, simple operation, and clean and harmless technology. Currently, there are many reported bacterial genera that can efficiently degrade pollutants, such as Pseudomonas, Flavobacterium, Corynebacterium, Achromobacter, Arthrobacter, Micrococcus, Vibrio, etc. There are almost no microorganisms in nature that can grow with cyclohexane as the sole carbon source. However, through laboratory domestication, screening, isolation, and culture, more and more microorganisms that can use cyclohexane as the sole carbon source have been reported. In 1919, Tausz and Pete first isolated a strain of Bacterium aliphaticumliquifaciens that can degrade cyclohexane. In 1948, Imelik isolated a strain of Pseudomonas aeruginosa that can degrade cyclohexane and studied its metabolites. In 1999, Li Danian et al. in China isolated a degradation strain that can use cyclohexane and cyclopentanone as the sole carbon source. After that, more and more reports on cyclohexane-degrading bacteria have emerged. Acinetobacter has been widely reported as one of the important microorganisms for degrading diesel.

[0004] The present invention screens out a novel Acinetobacter venetianus strain that uses cyclohexane as the sole carbon source from the environment, providing strong support for the microbial purification engineering application of soil, waste gas, and wastewater containing this pollutant. (3) Summary of the Invention

[0005] The object of the present invention is to provide a strain of Acinetobacter venetianus PFZR-1, which is a novel highly efficient cyclohexane-degrading bacterium with good environmental adaptability and can also effectively degrade other organic pollutants.

[0006] The technical solution adopted in the present invention is as follows:

[0007] The present invention provides a new strain for degrading cyclohexane, Acinetobacter venetianus PFZR-1, which is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M 2022719, the preservation date being May 25, 2022, and the address being Wuhan University, Wuhan, China, 430072.

[0008] The basic characteristics of the Acinetobacter venetianus PFZR-1 of the present invention are as follows: the colony is white, aerobic, smooth and moist, and ellipsoidal in shape.

[0009] The present invention also relates to the application of the Acinetobacter venetianus PFZR-1 in the microbial degradation of organic pollutants. The application is as follows: inoculating Acinetobacter venetianus PFZR-1 into an inorganic salt medium containing organic pollutants, and carrying out a degradation reaction under the conditions of pH = 5-10 (preferably pH = 6-8), 20-40 °C, and 100-200 rpm to achieve the degradation of organic pollutants; the organic pollutants include cyclohexane, toluene, methanol, limonene, ethyl acetate, cyclohexanol or cyclohexanone, preferably cyclohexane.

[0010] Preferably, the degradation reaction conditions are 30 °C and 160 rpm.

[0011] Preferably, the initial concentration of the organic pollutants in the inorganic salt medium is 63.3-316.4 mg / L (preferably 63.3-158.2 mg / L). The addition amount of the Acinetobacter venetianus PFZR-1 in the inorganic salt culture solution is based on the cell concentration OD 600 value and is 0.01-0.1, preferably 0.02.

[0012] The composition of the inorganic salt culture solution is as follows: 0.942 g / L of K2HPO4, 0.234 g / L of KH2PO4, 1.7 g / L of NaNO3, 0.98 g / L of NH4Cl, 0.2033 g / L of MgCl2·6H2O, 0.0111 g / L of CaCl2·2H2O, 0.0162 g / L of FeCl3, 5 ml / L of trace elements. The solvent is deionized water and the pH = 5 - 10; the composition of the trace elements is as follows: 0.088 g / L of ZnCl2, 0.060 g / L of MnCl2·4H2O, 0.01 g / L of KI, 0.1 g / L of Na2MoO4·2H2O, 0.05 g / L of H3BO3. The solvent is deionized water.

[0013] Before inoculation, Acinetobacter venetianus PFZR-1 of the present invention is first activated and subcultured, and the resting cells obtained by subculture are inoculated into the inorganic salt medium. The resting cells are prepared according to the following steps:

[0014] (1) Slant culture:

[0015] Acinetobacter venetianus PFZR-1 is inoculated into the LB solid medium and cultured in an incubator at 30 °C to obtain slant bacteria; the composition of the LB solid medium is: 5 g / L of yeast extract, 10 g / L of NaCl, 10 g / L of peptone, 15 - 20 g / L of agar, natural pH, and the solvent is deionized water;

[0016] (2) Subculture:

[0017] The slant bacteria obtained in step (1) are inoculated into the LB liquid medium and cultured at 30 °C and 160 rpm for 24 h to obtain a subculture solution. After centrifugation, the wet bacteria are collected and washed with the inorganic salt culture solution to obtain the resting cells of Acinetobacter venetianus PFZR-1; the composition of the LB liquid medium is: 5 g / L of yeast extract, 10 g / L of NaCl, 10 g / L of peptone, natural pH, and the solvent is deionized water.

[0018] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0019] The present invention provides a highly efficient degrading bacterium for organic pollutants, especially cyclohexane - Acinetobacter venetianus PFZR - 1. This bacterium is isolated from the sludge of a sewage treatment plant and can grow with cyclohexane as the sole carbon source and energy source. It can degrade cyclohexane at a concentration as high as 316.4 mg / L. Moreover, this bacterium has a wide suitable growth pH range and can degrade cyclohexane well at pH values from 6.0 to 8.0, with a degradation rate reaching over 90%. Acinetobacter venetianus PFZR - 1 has high degradation efficiency for cyclohexane, low cost, simple operation, and does not produce secondary pollution, having more advantages than traditional adsorption and photocatalytic degradation. Compared with existing cyclohexane - degrading bacteria, strain PFZR - 1 has a wide degradation concentration range (63.3 - 316.4 mg / L), strong adaptability (pH: 6.0 - 8.0), and can degrade a variety of other pollutants (toluene, methanol, limonene, ethyl acetate, cyclohexanol, and cyclohexanone). It provides strong technical support for the microbial purification engineering applications of soil, waste gas, and wastewater containing this pollutant. (IV) BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a photograph of the colony morphology of strain PFZR - 1 on LB medium.

[0021] Figure 2 It is a transmission electron microscope photograph of strain PFZR - 1.

[0022] Figure 3 It is the phylogenetic tree of strain PFZR - 1.

[0023] Figure 4 It is the degradation curve of strain PFZR - 1 for different concentrations of cyclohexane within 48 hours.

[0024] Figure 5 It is the degradation curve of strain PFZR - 1 for cyclohexane at different pH values within 48 hours. (V) SPECIFIC EMBODIMENTS

[0025] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0026] The following embodiments and drawings are only for illustrative purposes and should not be construed as limiting the present invention. Unless otherwise specified, the reagent raw materials used in the following embodiments are conventional commercially available or biochemistry reagent raw materials obtained through commercial channels, and the experimental instruments used are conventional laboratory instruments. Unless otherwise specified, the methods and equipment used in the following embodiments are the methods and equipment commonly used in the art.

[0027] The composition of the culture medium used in the embodiments of the present invention is as follows:

[0028] The composition of the inorganic salt culture medium is as follows: K2HPO4 0.942 g / L, KH2PO4 0.234 g / L, NaNO3 1.7 g / L, NH4Cl 0.98 g / L, MgCl2·6H2O 0.2033 g / L, CaCl2·2H2O 0.0111 g / L, FeCl3 0.0162 g / L, trace elements 5 ml / L. The solvent is deionized water, and the pH is 5 - 10. The composition of the trace elements is as follows: ZnCl2 0.088 g / L, MnCl2·4H2O 0.060 g / L, KI 0.01 g / L, Na2MoO4·2H2O 0.1 g / L, H3BO3 0.05 g / L. The solvent is deionized water.

[0029] The composition of the LB solid medium is: yeast extract 5 g / L, NaCl 10 g / L, peptone 10 g / L, agar 15 - 20 g / L, natural pH, and the solvent is deionized water.

[0030] The composition of the LB liquid medium is: yeast extract 5 g / L, NaCl 10 g / L, peptone 10 g / L, natural pH, and the solvent is deionized water.

[0031] Example 1: Isolation, purification and identification of Acinetobacter venetianus PFZR-1

[0032] 1. Sample collection and domestication

[0033] (1) Primary screening: Activated sludge collected from a domestic sewage treatment tank on-site was allowed to stand and settle for 2 h, then the supernatant and suspended impurities were removed, and the fine-grained sludge was left. The settled sludge at the bottom layer was mixed with the inorganic salt medium at a ratio of 1:1 (v / v) as a mixed medium and introduced into a sludge domestication tank with a total volume of 4 L. Using cyclohexane as the substrate as the sole carbon source and energy source, the sludge was domesticated at a constant temperature of (30 ± 1 °C). During the domestication experiment, the domestication tank was aerated stably, and the pH value of the domestication tank was controlled to maintain at 7.0. Every day, when the substrate concentration dropped below 30 mg / L, a substrate was added once to make the substrate concentration reach 100 mg / L after addition (the residual substrate concentration was detected by sampling before substrate addition). The mixed medium was replaced every 5 days. After nearly two months, the domesticated sludge could stably degrade 150 mg / L of cyclohexane every day. This sample was transferred to a shake flask for further enrichment to obtain a domesticated sample.

[0034] The above domesticated sample was inoculated into 50 mL of inorganic salt medium at an inoculation amount of 10% by volume. Using cyclohexane as the substrate with an initial concentration of 63.3 mg / L, it was cultured on a shaker at 30 °C and 160 r / min. The residual substrate concentration within 48 h was detected by the gas chromatography method described in Example 3. When the substrate degradation rate reached about 80%, the culture solution was transferred to a fresh inorganic salt medium containing cyclohexane at the same concentration (63.3 mg / L) at an inoculation amount of 10% by volume for subculture enrichment. Then, the initial concentration of cyclohexane (63.3 - 316.4 mg / L) was gradually increased for multiple subculture enrichments. The culture solution with a cyclohexane addition concentration of 316.4 mg / L was collected as the primary screening sample.

[0035] (2) Secondary screening: The primary screening samples were diluted and spread on an inorganic salt solid medium containing 63.3 mg / L cyclohexane. Using the inverted plate method, they were grown at 30 °C for 24 h, and single colonies were picked. After multiple streak separations of the single colonies on LB solid medium, they were then transferred to an inorganic salt medium with cyclohexane (initial concentration: 63.3 mg / L) as the sole carbon source and energy source and cultured at 30 °C for 48 h. The residual substrate concentration was tested by the gas chromatography method described in Example 3, and the degradation activity was calculated.

[0036] Single colonies with cyclohexane degradation ability were selected, and the secondary screening steps were repeated for further isolation and purification. When the degradation reached more than 80%, a strain with cyclohexane degradation activity was obtained and denoted as strain PFZR-1.

[0037] 2. Identification of strain PFZR-1

[0038] (1) Characteristics of strain PFZR-1:

[0039] The strain PFZR-1 was inoculated into LB medium and cultured at 30 °C for 24 h. The colonies were white, aerobic, smooth and moist, and ellipsoidal in shape. The colony morphology is shown in Figure 1 , and the transmission electron microscope photo is shown in Figure 2 .

[0040] (2) 16S rRNA sequence analysis

[0041] The DNA of strain PFZR-1 was extracted using PrepMan Ultra Kits nucleic acid extractant and stored at 4°C. Amplification was performed using universal bacterial primers (forward primer: 5’-AGAGTTTGATCCTGGCTCAG-3’; reverse primer: 5’-GGTTACCTTGTTACGACTT-3’) in a PCR amplifier from ABI. The nucleic acid sequencing of the purified PCR product was carried out using an Applied Biosystems 3500 genetic analyzer. The sequencing work was completed by Zhejiang Tianke High-Tech Development Co., Ltd. The 16S rDNA sequence of the strain is as follows (Genebank accession number: ON692913), SEQ ID NO.1:

[0042]

[0043] (3) Physiological and biochemical experiments

[0044] Utilization ability of strain ZRPF-1 for 46 carbon sources on the bioMérieux CBC card: The metabolic situation of the strain for 46 different carbon sources was investigated using a bioMérieux automatic identifier (entrusted to Zhejiang Tianke High-Tech Development Co., Ltd. (formerly Zhejiang Institute of Microbiology)). The identification results are shown in Table 1. Through the VITEK biochemical reaction of the bioMérieux automatic identifier, strain ZRPF-1 can strongly utilize 15 carbon sources and cannot utilize the other 31 carbon sources.

[0045] Table 1 VITEK biochemical reaction results of strain ZRPF-1 by bioMérieux automatic identifier (CBC card)

[0046]

[0047]

[0048] Table note: +, positive reaction; -: negative reaction

[0049] Through the research and analysis of the morphological characteristics and physiological and biochemical characteristics of strain PFZR-1, and at the same time comparing the sequence of this bacterium with the gene sequences in the NCBI database by Blast, a phylogenetic tree was constructed by combining the 16S RNA homology analysis (as Figure 3 ), so as to determine that strain PFZR-1 is Acinetobacter venetianus, named Acinetobacter venetianus PFZR-1, deposited in the China Center for Type Culture Collection, deposit number, CCTCC NO: M 2022719, deposit date, May 25, 2022, address: Wuhan University, Wuhan, China, 430072.

[0050] Example 2: Obtaining of resting cells of Acinetobacter venetianus PFZR-1

[0051] (1) Slant culture:

[0052] Inoculate Acinetobacter venetianus PFZR-1 into LB liquid medium and culture at 30 °C and 160 rpm for 48 h. Then streak the activated bacteria on an LB solid plate and culture in an incubator at 30 °C for 24 h. Take single colonies and continue to streak on the plate to detect the purity of the bacteria, and store them routinely in an LB test tube slant (4 °C).

[0053] (2) Subculture:

[0054] Inoculate the slant bacteria in step (1) into LB liquid medium, culture at 30 °C and 160 rpm for 24 h to obtain an enlarged culture solution, centrifuge, collect the wet bacteria, and wash with inorganic salt culture solution to obtain the resting cells of Acinetobacter venetianus PFZR-1.

[0055] Example 3: Detection of the degradation performance of Acinetobacter venetianus PFZR-1 on cyclohexane at different concentrations.

[0056] Under relatively suitable culture conditions (pH 7.0, temperature 30 °C), study the degradation of cyclohexane at different concentrations by Acinetobacter venetianus PFZR-1, specifically as follows:

[0057] Add different concentrations of the substrate cyclohexane to 50 mL of fresh inorganic salt medium, so that the initial concentrations of the substrate are 63.3, 94.9, 126.6, 158.2, 189.8, 253.12, 316.4 mg / L respectively. Inoculate the resting cells of Acinetobacter venetianus PFZR-1 prepared by the method of Example 2, so that the initial cell concentration in each parallel sample is 0.02 (in terms of OD 600 ). Culture in a shaker at 30 °C and a rotation speed of 160 r / min, sample regularly, determine the cyclohexane concentration by gas chromatography, and calculate the cyclohexane degradation rate of Acinetobacter venetianus PFZR-1. During the experiment, design 1 parallel sample and 1 blank control group without inoculating the strain. The results are as Figure 4 , Acinetobacter venetianus PFZR-1 can still completely degrade cyclohexane at a higher concentration (316.4 mg / L), but when the concentration of cyclohexane is greater than 316.4 mg / L and reaches 395.5 mg / L, it cannot be completely degraded within 60 hours.

[0058] Use a Fuli 9790II gas chromatograph to detect the concentration of cyclohexane in the gas phase. The chromatographic column is KB-5 (30 m × 0.32 mm × 0.5 μm). The injection port, detector (FID) and column temperature are 120 °C, 200 °C and 80 °C respectively, the auxiliary furnace temperature is 100 °C, and the split ratio is 100:1. The hydrogen flow rate is 30 mL / min, the air flow is 300 mL / min, the carrier gas nitrogen flow rate is 30 mL / min, and the gas injection volume is 1 mL.

[0059] Example 4: Detection of the degradation performance of Acinetobacter venetianus PFZR-1 on cyclohexane at different pH values.

[0060] Dispense 50 mL of inorganic salt culture solutions with pH values of 5, 6, 7, 8, 9, and 10 into shake flasks with a volume of 330 mL each, 50 mL per flask, and sterilize at 110 °C for 40 min. After sterilization, let it stand at room temperature for 2 d to confirm no growth of contaminants. Use cyclohexane as the sole carbon source for Acinetobacter venetianus PFZR-1, inoculate it into the inorganic salt medium, with an initial substrate concentration of 63.3 mg / L, and inoculate the resting cells of Acinetobacter venetianus PFZR-1 prepared by the method of Example 2 to make the initial cell concentration 0.02 (counted by OD 600 ), culture at a temperature of 30 °C and a rotation speed of 160 r / min, and set up a blank control without bacteria. Measure the residual cyclohexane concentration in the shake flask at regular intervals by the method of Example 3, and plot the removal rate curve of cyclohexane by the strain within 48 h at different pH values. The results are shown in Figure 5 . It shows that the optimal pH for Acinetobacter venetianus PFZR-1 to degrade cyclohexane is 7.

[0061] Example 5: Broad-spectrum degradation of substrates by Acinetobacter venetianus PFZR-1

[0062] In practical applications, there is not only cyclohexane as an organic pollutant. Industrial waste gases generally contain various volatile organic waste gases. Therefore, it is necessary to study the degradation effect of Acinetobacter venetianus PFZR-1 on other substrates. By the method of Example 3, change the carbon source to toluene, methanol, limonene, chloroform, ethyl acetate, cyclohexanol, and cyclohexanone with an initial concentration of 50 mg / L, and keep other operations the same as in Example 3. The degradation effects are shown in Table 2.

[0063] Table 2 Degradation effects of Acinetobacter venetianus PFZR-1 on different pollutants

[0064]

[0065] Table note: +, having degradation effect; -: having no degradation effect

[0066] As shown in Table 2: Acinetobacter venetianus PFZR-1 has different degrees of degradation effects on other selected pollutants except for chloroform. The reason is that chloroform is a halogenated hydrocarbon compound that is difficult to degrade, so the degradation effect is not good.

[0067] Although the present invention has been disclosed as above with examples, it is not intended to limit the protection scope of the present invention. Any modification and refinement made by those skilled in the art without departing from the concept and scope of the present invention shall fall within the protection scope of the present invention.

Claims

1. Acinetobacter venetianus( Acinetobacter venetianus ) PFZR-1, which is deposited in the China Center for Type Culture Collection, deposit number: CCTCC NO: M 2022719, deposit date: May 25, 2022, deposit address: Wuhan University, Wuhan, China.

2. Use of the Acinetobacter venetianus PFZR-1 according to claim 1 in microbial degradation of organic pollutants, characterized in that, The organic pollutant is cyclohexane, toluene, methanol, limonene, ethyl acetate, cyclohexanol or cyclohexanone.

3. The use according to claim 2, characterized in that, The application is as follows: inoculate Acinetobacter venetianus PFZR-1 into an inorganic salt medium containing the organic pollutant, and carry out a degradation reaction under the conditions of pH = 5-10, 20-40 °C, and 100-200 rpm to achieve the degradation of the organic pollutant.

4. The use according to claim 3, characterized in that, The initial concentration of cyclohexane in the inorganic salt medium is 63.3 - 316.4 mg / L; the addition amount of Acinetobacter venetianus PFZR-1 resting cells in the inorganic salt medium is based on the cell concentration OD 600 value of 0.01 - 0.

1.

5. The use according to claim 3, characterized in that, The composition of the inorganic salt medium is: 0.942 g / L of K2HPO4, 0.234 g / L of KH2PO4, 1.7 g / L of NaNO3, 0.98 g / L of NH4Cl, 0.2033 g / L of MgCl2·6H2O, 0.0111 g / L of CaCl2·2H2O, 0.0162 g / L of FeCl3, 5 ml / L of trace elements, the solvent is deionized water, and pH = 5-10; the composition of the trace elements is: 0.088 g / L of ZnCl2, 0.060 g / L of MnCl2·4H2O, 0.01 g / L of KI, 0.1 g / L of Na2MoO4·2H2O, 0.05 g / L of H3BO3, and the solvent is deionized water.

6. The use according to claim 3, characterized in that, Before inoculation, Acinetobacter venetianus PFZR-1 is first activated and expanded in culture, and the resting cells obtained by the expanded culture are inoculated into the inorganic salt medium. The resting cells are prepared according to the following steps: (1) Slant culture: Inoculate Acinetobacter venetianus PFZR-1 into an LB solid medium and culture it in an incubator at 30 °C to obtain slant bacteria; the composition of the LB solid medium is: 5 g / L of yeast extract, 10 g / L of NaCl, 10 g / L of peptone, 15-20 g / L of agar, natural pH, and the solvent is deionized water; (2) Expanded culture: Inoculate the slant bacteria in step (1) into an LB liquid medium and culture it at 30 °C and 160 rpm for 24 h to obtain an expanded culture solution. Centrifuge it to collect the wet bacteria, and wash them with an inorganic salt culture solution to obtain the resting cells of Acinetobacter venetianus PFZR-1; the composition of the LB liquid medium is: 5 g / L of yeast extract, 10 g / L of NaCl, 10 g / L of peptone, natural pH, and the solvent is deionized water.

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