Acinetobacter baumannii, bacterial agent and application thereof

Abstract

The application discloses a strain of Acinetobacter, the strain name is C16ZT1, and simultaneously proposes a bacterial agent containing the different bacillus, and further uses the bacterial agent for treating petroleum pollution wastewater. The Acinetobacter can efficiently degrade the petroleum production wastewater containing chain alkanes, can still realize the degradation effect quickly and stably under the conditions of high concentration, high salt and extreme acid and alkali, and can degrade more than 95% of the petroleum production wastewater within 144 hours. In the application in sewage treatment, the strain has strong tolerance, can tolerate high-concentration alkanes, and has a wide adaptation range to salt concentration, pH and the like, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology, specifically to an Acinetobacter bacillus for treating high-salt and alkaline petrochemical wastewater and its application in bioaugmentation treatment. Background Technology

[0002] The industrial production activities of petroleum exploration, extraction, transportation, and refining are indispensable to the increasing prosperity of modern industrial civilization. During the refining process of these products, a large amount of hydrocarbon-containing byproducts are inevitably generated. Compared with ordinary organic wastewater, petroleum fine chemical wastewater, represented by oil refineries, contains higher hydrocarbon concentrations, has complex compositions, poor water solubility, high salinity, and high toxicity. Furthermore, some cracking reactions produce strongly alkaline wastewater (pH>9), which inhibits the growth of common microorganisms, making its treatment even more difficult.

[0003] Compared to traditional physical and chemical remediation methods, microbial remediation has become one of the main methods for remediating petroleum pollution due to its economic and environmental advantages. Highly efficient microbial strains play a crucial role in this process. Currently, the screening of highly efficient petroleum-degrading microorganisms generally involves traditional microbiological methods, such as enrichment, separation, and purification. The high-purity bacterial agents obtained through cultivation exhibit high degradation efficiency for petroleum under laboratory conditions. However, the extreme environments in actual petrochemical wastewater, such as strong alkalinity (pH>9) and high salinity (>10g / L), can cause significant toxicity to these strains, making it difficult for them to adapt to environmental shocks and even leading to their death.

[0004] Total petroleum hydrocarbons (TPH) are an important pollutant indicator in environmental analysis and a persistent organic pollutant commonly found in petroleum-related pollution. Chain alkanes account for over 60% of TPH, and the environmental pollution they cause should not be underestimated.

[0005] The following are representative examples (laboratory studies) of Acinetobacter degradation of alkanes:

[0006] (1) The strain Acinetobacter sp. L12 (Wang Xinglong, et al. Screening and degradation characteristics of alkane-degrading bacteria in diesel-contaminated soil [J]. Modern Chemical Industry, 2022, 42(02): 163-166.) tolerated a salt concentration of no more than 30 g / L, with an optimal concentration of 0 g / L. It also tolerated a maximum pH of 10, with an optimal pH of 7. Under the optimal conditions, the degradation rate of C16 at 3000 mg / L was 83.93% within 7 days.

[0007] (2) The strain Acinetobacter haemolyticus.L1 (Li Yinghe, et al. Isolation, identification and degradation characteristics of hexadecane-degrading bacteria [J]. Journal of Harbin University of Commerce (Natural Science Edition), 2022, 38(02):131-136.) tolerated a salt concentration not exceeding 20 g / L, with an optimal concentration of 5 g / L. It also tolerated a maximum pH of 9, with an optimal pH of 8. Under optimal conditions, it achieved a degradation rate of 62.0% for 3867 mg / L C16 within 12 days.

[0008] (3) The strain Acinetobacter pittii SW-1 (Weina Kong, et al. Characterization and Transcriptome Analysis of a Long-Chain n-Alkane-Degrading Strain Acinetobacter pittii SW-1[J]. International Journal of Environmental Research and Public Health, 2021, 18(12).) tolerates a salt concentration of no more than 50 g / L, with an optimal concentration of 10 g / L. It also tolerates a maximum pH of 10, with an optimal pH of 8. Under optimal conditions, it has a degradation rate of 87.65% for 500 mg / L C20 within 7 days. Summary of the Invention

[0009] Purpose of the invention: To address the shortcomings of existing technologies, this invention provides an Acinetobacter bacterium capable of rapidly and stably degrading petrochemical wastewater even under high concentration, high salinity, and extremely acidic or alkaline conditions.

[0010] The technical problem to be solved by the present invention is to provide a bacterial agent prepared from the above-mentioned Acinetobacter and its preparation method.

[0011] Another technical problem to be solved by the present invention is to provide a bio-enhanced treatment technology for treating high-salinity petrochemical wastewater.

[0012] To address the first technical problem mentioned above, this invention discloses an Acinetobacter sp. strain named C16ZT1, which has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on October 12, 2022, with accession number CGMCC No. 25892.

[0013] Key biological characteristics of Acinetobacter: pale yellow, round, with regular edges, moist and smooth surface, raised center, and opaque cell. This strain forms round, raised colonies on nutrient agar plates with a smooth surface, regular edges, and opacity. The phylogenetic tree of this Acinetobacter strain is shown below. Figure 1 C.

[0014] The Acinetobacter sp. provided by this invention can grow using chain petroleum substances as the sole carbon source. When the initial concentration of chain petroleum substances is 10 g / L, inoculating with 5% volume of a bacterial agent prepared from Acinetobacter sp. results in a petroleum hydrocarbon degradation rate of over 85% after 144 hours.

[0015] Furthermore, the present invention discloses a bacterial agent comprising the aforementioned Acinetobacter.

[0016] The preparation method of the bacterial agent of the present invention is as follows: the Acinetobacter bacillus is activated on a culture medium, a single colony is picked and inoculated into LB medium, and cultured at 30-37℃ and 200 r / min to the logarithmic phase; then, it is inoculated into an inorganic salt culture medium at a volume ratio of 3%-5% and cultured at 30-37℃ and 200 r / min for 24 h to obtain the bacterial agent.

[0017] The LB culture medium comprises: 4.90–5.10 g / L yeast extract, 9.90–10.10 g / L peptone, and 4.90–5.10 g / L sodium chloride, with a pH of 7–8. Preferably, the LB culture medium comprises 5 g / L yeast extract, 10 g / L peptone, and 5 g / L sodium chloride, with a pH of 7–8.

[0018] The inorganic salt culture medium comprises: NaCl 0.95–1.05 g / L, (NH4)2SO4 0.95–1.05 g / L, KH2PO4 0.48–0.52 g / L, K2HPO4 1.48–1.52 g / L, MgSO4·7H2O 0.19–0.21 g / L, pH 7.0–8.0. Preferably, the inorganic salt culture medium comprises: NaCl 1.0 g / L, (NH4)2SO4 1.0 g / L, KH2PO4 0.5 g / L, K2HPO4 1.5 g / L, MgSO4·7H2O 0.2 g / L, pH 7–8.

[0019] For inorganic salt culture media where the composition is not fixed, the following inorganic salt culture media can also be used: (NH4)2SO4 1.48~1.52g / L, KH2PO4 0.49~0.51g / L, K2HPO4·3H2O 1.90~1.93g / L, NaCl 0.48~0.52g / L, MgSO4·7H2O 0.19~0.21g / L, pH 6.8~7.2; or K2HPO4·3H2O 0.98~1.02g / L, KH2PO4 0.98~1.02g / L, MgSO4·7H2O 0.48~0.52g / L, NH4NO3 0.98~1.02g / L, CaCl2 0.018~0.022g / L, Fe2(SO4)3 0.018~0.022g / L, pH 7.0~7.2.

[0020] The present invention also discloses the application of the above-mentioned Acinetobacter or bacterial agent in the degradation of chain alkanes, wherein the chain alkanes are chain alkanes with a chain length of no more than 40 carbon atoms.

[0021] Furthermore, this invention discloses the application of the aforementioned Acinetobacter or bacterial agent in treating high-salt, highly alkaline petrochemical wastewater, wherein the petrochemical wastewater has a pH of 3-13 and a salt content not exceeding 100 g / L. In some embodiments, the sodium chloride content in the high-salt petrochemical wastewater is 1-5 g / L.

[0022] During treatment, the Acinetobacter bacillus or bacterial agent is inoculated into the petrochemical wastewater to be treated at a volume ratio of 1% to 10%.

[0023] In some embodiments, the degradation temperature is 15–40°C; in some embodiments, the degradation temperature is 30–32°C.

[0024] In some embodiments, the total content of chain alkanes in the high-salt chemical wastewater is 0.05–10 g / L; in some embodiments, the total hydrocarbon content in the high-salt chemical wastewater is approximately 2 g / L.

[0025] In some embodiments, the chain alkane is total petroleum hydrocarbon (C8 to C40) with a chain length of less than 40 carbon atoms.

[0026] In some embodiments, the chain alkane is n-hexadecane. n-Hexadecane, i.e., C16, represents the C10–C40 portion of the chain alkane. Since chain alkanes are structurally similar, they can be considered as a single entity in existing studies, similar to normalization. In existing articles, there are numerous studies and case studies using C16 as the research object to screen petroleum hydrocarbon-degrading bacteria and to study the transformation and fate of petroleum hydrocarbons in the environment. This indicates that the practice of using C16 as a typical representative of C10–C40 hydrocarbons is widely accepted, and selecting it as a representative substance aligns with mainstream practices.

[0027] Beneficial Effects: This invention targets hexadecane (a chain alkane model pollutant) and obtains a highly efficient alkane-resistant strain tolerant to high salt and strong alkali. By adding the Acinetobacter agent to hydrocarbon-containing chemical production wastewater using bioaugmentation technology, it can achieve a high-efficiency degradation of over 95% within 144 hours. The optimal operating temperature is 15–40℃, pH is 3–13, and salt concentration is 0–100 g / L (calculated as sodium chloride). In wastewater treatment applications, this strain demonstrates strong tolerance, can withstand high concentrations of mixed hydrocarbons, and exhibits a wide range of adaptability to varying salt concentrations and pH conditions, showing promising application prospects. This invention provides theoretical and technical support for the application of highly efficient petroleum-degrading microorganisms and offers a new microbial resource for the bioremediation of petroleum-polluted environments.

[0028] Figure and Table Description

[0029] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention as described above or otherwise will become clearer.

[0030] Figure 1 The colony morphology of Acinetobacter sp. C16ZT1 is shown in (A) and scanning electron microscope (B), and the phylogenetic tree is shown in (C).

[0031] Figure 2 The graph shows the degradation rate of alkanes by Acinetobacter at different concentrations.

[0032] Figure 3 The effect of pH on the degradation efficiency of Acinetobacter;

[0033] Figure 4 The effect of salt concentration (calculated as sodium chloride) on the degradation efficiency of Acinetobacter;

[0034] Figure 5 The gas chromatogram shows the C10–C40 content of the wastewater at the initial moment of the degradation experiment.

[0035] Figure 6 The curves showing the change of alkane residual concentration over time in experiments using Acinetobacter sp. to treat actual mixed chain hydrocarbon (C10-C40) wastewater.

[0036] Figure 7 The chromatogram shows the C10 to C40 content of the wastewater after the degradation experiment was completed. Detailed Implementation

[0037] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0038] Unless otherwise specified, the inoculation amounts described in the following examples are all based on the corresponding volume ratios.

[0039] In the following examples, the content of alkane was determined using gas chromatography (with slight modifications based on national standards and literature), as detailed below:

[0040] ① Transfer the fermentation broth to be analyzed into a brown glass bottle. Add a certain volume of chromatographically pure hexane to wash the shake flask, and combine the washing solution with the brown glass bottle. Vortex at 2000 rpm for 10 min, then extract ultrasonically (temperature: 30℃, ultrasonic frequency: 40 kHz) for 30 min. Finally, centrifuge at 3000 rpm for 5 min to separate the organic and aqueous phases. Repeat the extraction three times. Collect and combine the upper organic phases and filter them through a 0.22 μm filter membrane for gas chromatography analysis.

[0041] ②The specific settings for gas chromatography are as follows: RB-5 quartz capillary column (30m×0.32mm×0.25μm, 5% phenyl, 95% methyl polysiloxane), FID detector, carrier gas is high-purity nitrogen, controlled flow rate is 30mL / min, hydrogen flow rate is 30mL / min, air flow rate is 300mL / min, the inlet and detector temperatures are both 300℃, the injection volume is 1.0μL, and the column oven temperature program is as follows: hold at 40℃ for 1.0min, increase to 290℃ at 15℃ / min, and hold for 1min.

[0042] ③ The external standard method was used to quantitatively analyze the concentration of alkanes in the fermentation broth. The standard curve for this experiment was y = 0.0015x - 7.334, R0. 2 =0.9998.

[0043] Example 1: Isolation, purification, and identification of the strain.

[0044] 10 mL of activated sludge was added to an inorganic salt culture medium containing 1 g / L hexadecane. The activated sludge was obtained from a biochemical tank in Jiangsu Province that has been treating petroleum refining wastewater for a long time. Enrichment culture was carried out at 30℃ and 200 r / min. The inoculum was then transferred to a new inorganic salt liquid culture medium containing hexadecane at a rate of 10%, and this process was repeated three times to obtain the enriched solution. The inorganic salt culture medium consisted of: NaCl 1.0 g / L, (NH4)2SO4 1.0 g / L, KH2PO4 0.5 g / L, K2HPO4 1.5 g / L, MgSO4·7H2O 0.2 g / L, and pH 7–8.

[0045] The enriched solution was serially diluted, and 10... -5 0.1 mL of the diluted liquid was spread onto a solid culture medium containing n-hexadecane. Single colonies were picked, isolated, and purified to obtain pure bacteria. These pure bacteria were then inoculated into liquid culture to verify their ability to degrade alkane compounds. Finally, a strain capable of efficiently degrading alkane compounds was obtained, such as... Figure 1 As shown in Figure A. It was identified as Acinetobacter sp. C16ZT1, and the scanning electron microscope image of the strain is shown below. Figure 1 As shown in B.

[0046] The solid culture medium containing n-hexadecane consists of: NaCl 1.0 g / L, (NH4)2SO4 1.0 g / L, KH2PO4 0.5 g / L, K2HPO4 1.5 g / L, MgSO4·7H2O 0.2 g / L, agar 2%, pH 7-8, with a certain amount of n-hexadecane added before pouring the plates.

[0047] After extracting DNA from the purified strain, PCR amplification was performed using the universal bacterial primer 27F-1492R, followed by 16S rDNA sequencing. The alignment result identified it as *Acinetobacter sp.*, named C16ZT1. The obtained strain sequence was entered into the EZBioCloud database for BLAST alignment. Multiple sequence alignment and analysis were performed on the most similar strain sequences using MEGA7 software. A phylogenetic tree was constructed using Neighbour Joining (NJ) as follows: Figure 1 As shown in Figure C. Its main physiological characteristics are Gram-negative staining, forming round, raised colonies on nutrient agar plates, pale yellow in color, with neat edges, a moist and smooth surface, a raised center, and opaque cells. Scanning electron microscopy of a small sample revealed coccobacilli-shaped cells with an average diameter of approximately 1.2–2 μm. Physiological and biochemical identification of the strain was performed according to the *Handbook of Systematic Identification of Common Bacteria*, and the results are shown in Table 1.

[0048] Table 1

[0049]

[0050]

[0051] Example 2: Preparation of bacterial agent.

[0052] The strain C16ZT1 was activated on LB medium, and a single colony was picked and inoculated into LB medium. The culture was then shaken at 30-32℃ and 200 rpm until the logarithmic phase. The strain was then inoculated into inorganic salt medium at a rate of 5% (V / V) and shaken at 30℃ and 200 rpm to obtain the corresponding bacterial agent.

[0053] The LB medium consists of: 5 g / L yeast extract, 10 g / L peptone, and 5 g / L sodium chloride, with a pH of 7–8.

[0054] The inorganic salt culture medium consists of: NaCl 1.0 g / L, (NH4)2SO4 1.0 g / L, KH2PO4 0.5 g / L, K2HPO4 1.5 g / L, MgSO4·7H2O 0.2 g / L, pH 7–8.

[0055] Example 3: Degradation experiment of hydrocarbons by strain C16ZT1.

[0056] Acinetobacter bacillus strain C16ZT1, cultured to the logarithmic growth phase in culture medium, was inoculated at a volume ratio of 5% into a medium containing different concentrations of the chain alkane n-hexadecane. The medium composition was NaCl 1.0 g / L, (NH4)2SO4 1.0 g / L, KH2PO4 0.5 g / L, K2HPO4 1.5 g / L, MgSO4·7H2O 0.2 g / L, pH 7–8. The medium was cultured in a shaker at 30–32 °C and 200 rpm. The alkane degradation efficiency was measured after 144 h. Figure 2 As shown, Acinetobacter's degradation efficiencies for alkanes at initial concentrations of 0.05 g / L, 0.5 g / L, 1 g / L, 2 g / L, 5 g / L, and 10 g / L were 92%, 95%, 100%, 98%, 92%, and 87%, respectively.

[0057] Example 4: Effect of pH on degradation efficiency.

[0058] Acinetobacter bacillus strain C16ZT1, cultured to the logarithmic growth phase in culture medium, was inoculated at a volume ratio of 5% into inorganic salt culture media with different initial pH values: 3, 5, 7, 9, 11, and 13 (the pH was adjusted using 1 mol / L hydrochloric acid and sodium hydroxide). The culture medium composition was: NaCl 1.0 g / L, (NH4)2SO4 1.0 g / L, KH2PO4 0.5 g / L, K2HPO4 1.5 g / L, MgSO4·7H2O 0.2 g / L, pH 7–8. The alkane degradation rate was measured after 144 hours. Figure 3 As shown, the degradation rate of alkanes was above 95% within a pH range >7, indicating that this strain has high adaptability to alkaline wastewater.

[0059] Example 5: Effect of salt concentration on degradation efficiency.

[0060] Acinetobacter bacillus strain C16ZT1, cultured to the logarithmic growth phase in a culture medium, was inoculated at a 5% inoculum into media containing different salinities (calculated as sodium chloride). The media contained 1.0 g / L NaCl, 1.0 g / L (NH4)2SO4, 0.5 g / L KH2PO4, 1.5 g / L K2HPO4, and 0.2 g / L MgSO4·7H2O, with a pH of 7–8. The sodium chloride concentration was adjusted to 0 g / L, 5 g / L, 10 g / L, 20 g / L, 50 g / L, and 100 g / L, respectively. The media were cultured at 30–32 °C in a shaker at 200 rpm for 144 h. The alkane content was measured, and the degradation rate was as follows: Figure 4 As shown, Acinetobacter's degradation rate of alkane was above 80% in the salt concentration range of 0 g / L to 100 g / L; these results indicate that this strain has high tolerance to wastewater salt concentration.

[0061] Example 6: Effect of Acinetobacter C16ZT1 on the treatment of total petroleum hydrocarbons (C10-C40) in petrochemical wastewater.

[0062] The gas chromatogram of C10-C40 content in the wastewater at the initial moment of the degradation experiment is shown below. Figure 5 As shown.

[0063] Acinetobacter C16ZT1 bacterial suspension (cultured to the logarithmic phase and resuspended in sterile water on LB medium, 30–37℃, 200 rpm, logarithmic phase 6–8 h, cell OD value 1.0–1.5) was inoculated at a volume ratio of 5% into the production wastewater of an oil refinery in Jiangsu Province. The original wastewater contained approximately 2 g / L of total petroleum hydrocarbons. Wastewater parameters were: pH = 10.74 ± 1.34, salinity = 2.63 ± 0.97 g / L. The wastewater was cultured in a shaker at 30–32℃, 200 rpm. The total petroleum hydrocarbon content of the inoculated and uninoculated wastewater systems was measured after 144 h. The results are as follows: Figure 5 As shown, compared with the uninoculated wastewater, the group treated with strain C16ZT1 showed a significant increase in hydrocarbon degradation. The chromatograms of C10–C40 content in the wastewater after the degradation experiment are shown below. Figure 7 As shown, most of the hydrocarbons have been degraded.

[0064] This invention provides a concept and method for bio-enhanced treatment of high-salt, highly alkaline petrochemical wastewater. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A strain of Acinetobacter, classified as Acinetobacter (… Acinetobacter The strain, named C16ZT1, has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 12, 2022, with accession number CGMCC No. 25892.

2. An inoculant characterized in that, The Acinetobacter according to claim 1.

3. The method for preparing the bacterial agent of claim 2, characterized by, The Acinetobacter according to claim 1 is activated and inoculated into LB medium, and cultured at 30-37 DEG C, 200-300 r / min to logarithmic phase; then inoculated into inorganic salt medium at 3%-5% of volume ratio, and cultured at 30-37 DEG C, 200-300 r / min, to obtain the microbial inoculum.

4. The production method according to claim 3, characterized by, The LB medium comprises 4.90-5.10 g / L of yeast powder, 9.90-10.10 g / L of peptone, and 4.90-5.10 g / L of sodium chloride, and has a pH of 7-8; and the inorganic salt medium comprises 0.95-1.05 g / L of NaCl, 0.95-1.05 g / L of (NH4)2SO4, 0.48-0.52 g / L of KH2PO4, 1.48-1.52 g / L of K2HPO4, and 0.19-0.21 g / L of MgSO4·7H2O, and has a pH of 7.0-8.

0.

5. Use of the Acinetobacter bacterium of claim 1 or the bacterial agent of claim 2 for degrading chain-alkanes, wherein, The chain alkane substance has a chain length of not more than 40 carbon atoms.

6. The use of Acinetobacter as claimed in claim 1 or the bacterial agent as claimed in claim 2 for treating high-salinity, strong-alkalinity petrochemical wastewater, wherein, The petroleum chemical wastewater has a pH of 3-13 and a salt content of not higher than 100 g / L.

7. Use according to claim 6, characterized in that, During the treatment, the Acinetobacter according to claim 1 or the microbial inoculum according to claim 2 is inoculated into the petroleum chemical wastewater to be treated at 1%-10% of volume ratio.

8. Use according to claim 6, characterized in that, During the treatment, the degradation temperature is 15-40 DEG C.

9. Use according to claim 6, characterized in that, The petroleum chemical wastewater contains the chain alkane substance at a content of 0.05-10 g / L.

10. Use according to claim 6, characterized in that, The petroleum chemical wastewater contains the chain alkane substance having a chain length of not more than 40 carbon atoms.

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