CN103695351B - Acinetobacter baumannii and application thereof - Google Patents

Abstract

The invention discloses an Acinetobacter baumannii and its application. The Acinetobacter baumannii of the present invention is named Acinetobacter baumannii DD1 (Acinetobacter baumannii DD1), and the preservation number is CCTCC NO: M2013560, which was registered in November 2013 On the 8th, it was preserved in the Chinese Type Culture Collection Center located in Wuhan University, Wuhan, China. Inoculating the Acinetobacter baumannii into dioxane-containing wastewater or an inorganic salt medium fed with dioxane waste gas can achieve complete degradation of dioxane, and can also degrade pollution such as toluene, phenol, tetrahydrofuran, etc. things. The bacterial strain of the present invention is an aerobic non-fermentative Gram-negative bacterium, which can efficiently degrade the substrate while growing with dioxane as the only carbon source and energy source; the bacterial strain can degrade various pollutants such as toluene, phenol, and tetrahydrofuran; The invention lays a foundation for the engineering application of purifying waste water and waste gas containing dioxane by biological method.

Description

A kind of Acinetobacter baumannii and application thereof

technical field

The invention relates to a method for degrading dioxane, in particular to Acinetobacter baumannii and its application.

Background technique

1,4-dioxane (1,4-dioxane), also known as dioxane, has good water solubility and is a good organic solvent, compatible with ethanol, ether, acetone, phenol, etc. , are widely used in industries such as paints, dyes, and pharmaceuticals, and are also used in consumer products such as food, cosmetics, and detergents. Dioxane is irritating to the skin, eyes and respiratory system, and can accumulate in the human body, causing serious damage to the liver, kidney and nervous system. Chronic cumulative poisoning can lead to uremia, kidney failure and other diseases, and acute poisoning may cause Death, classified as Class B2 (probable) human carcinogen by the U.S. Environmental Protection Agency (U.S.EPA) and the World Health Organization International Cancer Research Center (IARC).

The extensive application of dioxane has resulted in increasingly serious pollution of surface water and groundwater. At present, dioxane pollution has been detected in groundwater and landfills in many developed countries such as the United States, Canada, and Japan; high concentrations have been detected in groundwater in Ottawa, Canada, Lake Ontario, and landfills near Canada Dioxane: A large amount of dioxane was also detected in the Yellow River of my country, and the accumulation of dioxane was also detected in the viscera of indigenous fish in the Lanzhou section. More studies have shown that dioxane has been detected even in the uninhabited Arctic permafrost and groundwater. Therefore, the removal of dioxane in surface water and groundwater is urgent, and an effective method is urgently needed to remove it.

At present, the methods for removing dioxane at home and abroad are mainly chemical oxidation methods, including ozone, UV light, hydrogen peroxide, and Fenton oxidation. These methods have their own advantages, but such methods are too expensive to apply Environmental pollution control of dioxane. Due to its physical and chemical properties such as cyclic ether structure, C-O high-energy bond, low Henry constant and low octanol-water partition coefficient, dioxane was once classified as a "non-biodegradable substance".

In 1991, German scholar Bemhardt et al. isolated the strain Rhodococcus ruber219 with dioxane degradation ability from the sludge of dioxane chemical plant for the first time, realizing the biodegradation of dioxane for the first time. Thus, more and more researchers began to pay attention to the research of biological treatment of dioxane. In 1993, Burback et al. isolated a strain Mycobacterium vaccae with dioxane-degrading ability, but the strain had limited dioxane-degrading ability and could not grow continuously. Mycobacterium PH-06 reported by Young-Mo Kim et al. can degrade 90% of 1000mg/L dioxane within 15 days, but the degradation cycle is too long. The fungus Cordyceps sinensis isolated by Nakamiya et al. can also use dioxane as the only carbon and energy source for sustainable growth. Currently reported dioxane-degrading bacteria include Pseudonocardia dioxanivorans CB1190, Pseudonocardia B5, Bacillus pumilus D4 and Xanthobacter D7.

Contents of the invention

The invention provides an Acinetobacter baumannii and its application. The Acinetobacter baumannii can degrade dioxane at a rate above 99.9%, and can also degrade toluene, phenol, tetrahydrofuran, etc. pollutants.

A kind of Acinetobacter baumannii named Acinetobacter baumannii DD1 (Acinetobacter baumannii DD1), and the preservation number is CCTCC NO: M2013560.

Acinetobacter baumannii DD1 (Acinetobacter baumannii DD1) of the present invention was preserved on November 8, 2013 in the Chinese Type Culture Collection Center located in Wuhan University, Wuhan, China.

The Acinetobacter baumannii DD1 of the present invention is collected from the activated sludge of the secondary sedimentation tank of the sewage treatment plant, has a short rod shape, a size of 1.0～1.5×1.5～2.5 μm, no flagella, no spores; After culturing at 30°C for 48 hours, the single colony was spherical, smooth and moist, and the bacterial lawn grew along the streaked line; Gram staining was negative, and oxidase and contact enzyme tests were negative; gelatin, citrate, and nitrate reduction tests were positive ; It is resistant to kanamycin and rifamycin, but not to tetracycline; it grows slowly when the salinity is greater than 4%.

The present invention also provides an application of the Acinetobacter baumannii in degrading dioxane.

The application is specifically: inoculate the Acinetobacter baumannii into wastewater containing dioxane, inorganic salt medium containing dioxane or inorganic salt medium with dioxane exhaust gas for cultivation, to degrade dioxane alkyl.

The Acinetobacter baumannii DD1 (Acinetobacter baumannii DD1) can use dioxane as the sole carbon source and energy source for growth and reproduction, and mineralize dioxane into CO ₂ and H ₂ O. Under pure culture conditions, the bacteria can completely degrade 100mg/L dioxane in the inorganic salt medium within 42 hours.

More preferably, the cultivation is carried out at a pH value of 5.0-8.0 and a temperature of 25°C to 40°C. More preferably, it carries out in the range of pH 7.0 and temperature 32 degreeC.

Further preferably, the initial concentration of dioxane in the wastewater is 100 mg/L to 500 mg/L; the initial concentration of dioxane in the inorganic salt medium containing dioxane is 100 mg/L to 500 mg/L; The initial concentration of dioxane in the inorganic salt medium through which dioxane waste gas is introduced is 100 mg/L-500 mg/L. More preferably both are 200mg/L.

The initial concentration of dioxane in the inorganic salt medium that is fed with dioxane waste gas is 100mg/L～500mg/L means: the initial concentration of dioxane in the inorganic salt medium refers to the dioxane that is passed into The initial concentration of dioxane dissolved in the inorganic salt culture medium in the exhaust gas, therefore, the feed amount of the dioxane exhaust gas is measured by the required initial concentration of dioxane in the inorganic salt medium.

More preferably, the culture time is 20 to 50 hours, more preferably 42 hours.

Further preferably, the inorganic salt medium (MSM) contains the following substances: 3.5g Na ₂ HPO ₄ ·2H ₂ O, 1.0g KH ₂ PO ₄ , 0.5g (NH ₄ ) ₂ SO ₄ , 0.1g MgCl ₂ ·6H ₂ O, 0.05g Ca(NO ₃ ) ₂ , dissolved in 1000mL water, added 1ml trace element solution, adjusted pH to 7.0-7.2. The medium components of the inorganic salt medium containing dioxane or the inorganic salt medium blown with dioxane waste gas are partly the same.

The composition of the trace element solution is FeSO ₄ 7H ₂ O 1.0g, CuSO ₄ 5H ₂ O 0.02g, H ₃ BO ₃ 0.014g, MnSO ₄ 4H ₂ O 0.10g, ZnSO ₄ 7H ₂ O 0.10g, Na ₂ MoO ₄ ·2H ₂ O0.02g, _CoCl2 · _6H2O0.02g , dissolved in 1000mL of water.

The Acinetobacter baumannii of the present invention can also be used to degrade phenol, toluene, tetrahydrofuran or o-xylene.

The Acinetobacter baumannii is inoculated into an inorganic salt medium containing phenol, toluene or tetrahydrofuran for culture to degrade the phenol, toluene or tetrahydrofuran. Other degradation conditions are the same as degrading dioxane, and the degradation time is different. The degradation rate of bacterial strain of the present invention to phenol degradation 20h reaches 99.9%, the degradation rate of toluene degradation 24h reaches 50%, and the degradation rate of tetrahydrofuran degradation 48h reaches 99.9%. 99.9%.

The Acinetobacter baumannii of the present invention can also be used to degrade n-butanol, n-hexane, ethanol or benzene, and the degradation rate can reach 99.9%. The method of degradation treatment is the same as the degradation of dioxane, and the degradation time is different. The optimal degradation time of n-butanol is 24h, the optimal degradation time of n-hexane is 108h, the optimal degradation time of ethanol is 24h, and the optimal degradation time of benzene is 24h. The best degradation time is 24h.

The beneficial effects of the present invention are:

The invention provides a strain of Acinetobacter baumannii capable of efficiently degrading dioxane. The strain is an aerobic non-fermenting Gram-negative bacterium, which can efficiently degrade the dioxane while growing with dioxane as the only carbon source and energy source. The substrate has a degradation rate of more than 99%; the strain can degrade various pollutants such as toluene, phenol, tetrahydrofuran, etc.; the invention lays a foundation for the engineering application of purifying dioxane-containing wastewater or waste gas by biological methods.

Description of drawings

Fig. 1 is Acinetobacter baumannii DD1 degrading dioxane and bacterial cell growth;

Fig. 2 is the influence of different pH on bacterium concentration and dioxane degradation rate;

Fig. 3 is the change of dioxane concentration with time under different temperatures;

Fig. 4 is the change of dioxane concentration with time under different initial dioxane concentrations;

Figure 5 is the degradation of phenol by Acinetobacter baumannii DD1 and the growth of bacteria;

Figure 6 shows the degradation of THF by Acinetobacter baumannii DD1 and the growth of the bacteria.

Detailed ways

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

Mineral salt medium (MSM) contains the following substances: 3.5g Na ₂ HPO ₄ 2H ₂ O, 1.0g KH ₂ PO ₄ , 0.5g (NH ₄ ) ₂ SO ₄ , 0.1g MgCl ₂ 6H ₂ O, 0.05g Ca (NO ₃ ) ₂ , dissolved in 1000mL of water, added 1ml of trace element solution, and adjusted to pH 7.0-7.2.

The composition _of trace element solution is FeSO ₄ ·7H ₂ O1.0g, _CuSO4 · _5H2O0.02g , _H3BO30.014g , _MnSO4 · _4H2O0.10g , ZnSO4 _{· 7H2O0.10g} , _Na2 MoO ₄ ·2H ₂ O 0.02g, CoCl ₂ ·6H ₂ O 0.02g, dissolved in 1000mL of water.

Embodiment 1: Isolation and identification of Acinetobacter baumannii DD1

(1) Sample collection and domestication

The activated sludge from the secondary sedimentation tank of Hangzhou Qige Wastewater Treatment Plant was collected on site, and dioxane was used as the only carbon source and energy source for domestication and enrichment. Several months later, the enriched activated sludge was inoculated into a 250mL conical flask containing 100mL MSM medium, and dioxane was used as the only carbon source and energy source to continue the cultivation and enrichment. The experiment needs to be kept at constant temperature (30±1°C) and kept under aerobic conditions.

(2) Strain isolation and identification

The mixed bacterial solution enriched through multiple passages was serially diluted, and inoculated on the inorganic salt agar medium with dioxane as the only carbon source by coating method, and placed in a constant temperature incubator (30°C) nourish. Pick single colonies. After the single colony was separated by streaking several times, it was cultured in the MSM medium containing 100mg/L dioxane) to test whether it had the ability to degrade dioxane. The strains with degradation ability were selected for further isolation and purification until the strain DD1 with dioxane degradation ability was isolated.

The shape of the strain is short rod, the size is 1.0～1.5×1.5～2.5μm, no flagella, no spores; after cultured on solid plate medium at 30℃ for 48h, the single colony is spherical, light yellow, smooth and moist, with a lawn along the Streak growth; negative Gram staining; positive for gelatin, citrate, and nitrate reduction tests; negative for oxidase and catalase tests; resistant to kanamycin and rifamycin, no to tetracycline Resistance; growth is slow when salinity is greater than 4%.

The above characteristics are consistent with the physiological and biochemical characteristics of Acinetobacter recorded in the "Bergery Bacteria Identification Manual". The strain was identified as Acinetobacter baumannii by Biolog microbial identification and 16S rDNA homology analysis, combined with the above physiological and biochemical bacteriological characteristics, the full gene sequence of 16S rDNA is shown in SEQ ID NO: 1, GenBank The accession number is KF713537.

The Acinetobacter baumannii was named as Acinetobacter baumannii DD1 (Acinetobacter baumanniiDD1), and it was preserved in the Chinese Type Culture Collection Center located in Wuhan University, Wuhan, China on November 8, 2013, with the preservation number CCTCC NO : M2013560.

Embodiment 2: the characteristic of Acinetobacter baumannii DD1 degradation dioxane

(1) Dioxane is used as the sole carbon source of Acinetobacter baumannii DD1, inoculate Acinetobacter baumannii DD1 cells into 100mL inorganic salt medium, the initial cell OD ₆₀₀ is 0.01; add dioxane to make the initial dioxane concentration 100mg/ L. Place them in a shaker with a temperature of 30°C and a rotation speed of 130r/min, and take samples regularly. After 42 hours of cultivation, the OD value of the strain reaches a maximum value of 0.183. The results are shown in Figure 1.

It can be seen from Figure 1 that with the prolongation of time, the concentration of bacteria species also increased. This example shows that the degrading bacteria Acinetobacter baumanniiDD1 can use dioxane as the only carbon source and energy source for biological reproduction, and has the ability to efficiently degrade dioxane.

(2) Use NaOH or HCl solution to adjust the inorganic salt medium to different pH values (5.0, 6.0, 6.5, 7.0, 7.5, 8.0). Under the condition that the initial dioxane concentration is 100 mg/L, the strains are inserted into the The initial cell OD ₆₀₀ was 0.01. The samples were shaken and cultivated in a constant temperature shaker at 30°C and 130r/min, and samples were taken after 35 hours of cultivation. The results are shown in Figure 2.

It can be seen from Figure 2 that within the pH range of 5.0 to 8.0, microorganisms can degrade dioxane well, and with the increase of cell concentration; as the pH continues to increase, the strain concentration and dioxane degradation first increase The optimum pH for degradation is 7.0.

(3) In the inorganic salt medium with an initial dioxane concentration of 100 mg/L, insert the strain so that the initial cell OD ₆₀₀ is 0.01. Set the temperature of each sample to 25°C, 30°C, 32°C, 37°C, and 40°C, place them in a 130r/min constant temperature shaker for shaking culture, take samples regularly, and measure the residual concentration of dioxane. The results are shown in Figure 3 . It can be seen from Figure 3 that the strains can grow in the range of 25°C to 40°C, and the degradation rate has a process of first increasing and then decreasing with the increase of temperature, and the degradation rate reaches the maximum at 32°C.

(4) In the inorganic salt medium with the initial dioxane concentration of 100mg/L, 200mg/L, 300mg/L, 400mg/L, and 500mg/L, insert the strain so that the initial cell OD ₆₀₀ is 0.01, and the The samples were shaken and cultured in a constant temperature shaker at 30°C and 130r/min, and samples were taken regularly. The results are shown in Figure 4. It can be seen from Figure 4 that, except for 500mg/L, they can be completely degraded, and the degradation rate has a process of first increasing and then decreasing with the increase of concentration, and the degradation rate is the fastest in the case of 200mg/L.

Embodiment 3: Acinetobacter baumannii DD1 degrades phenol and tetrahydrofuran

Using phenol and tetrahydrofuran as the sole carbon source and energy source of Acinetobacter baumannii DD1 respectively, in the inorganic salt medium with an initial concentration of 100 mg/L, insert the strain so that the initial cell OD ₆₀₀ is 0.01, and place the sample at 30°C, Vibration cultivation in a 130r/min constant temperature shaker, regular sampling, the results are shown in Figure 5 and Figure 6.

From the results in Figure 5, it can be seen that with the prolongation of the culture time, the concentration of the bacteria increases and the concentration of phenol decreases. When the culture time exceeds 20 h, the phenol is basically completely degraded;

From the results in Figure 6, it can be seen that with the prolongation of the culture time, the concentration of bacteria increases and the concentration of tetrahydrofuran decreases. When the culture time reaches 70h, tetrahydrofuran is basically completely degraded;

Embodiment 4: Acinetobacter baumannii DD1 substrate broad-spectrum degradation situation

In the inorganic salt medium, add various substrates respectively (that is, the substrates in Table 1 are used as the single carbon source), and the dosage is shown in Table ₁ below. The samples were shaken and cultivated in a constant temperature shaker at 30°C and 130r/min, and samples were taken regularly for measurement. The results showed that the strains could not directly utilize pollutants such as chloroform, trichloroethylene, trichloroacetic acid, isooctane, etc. The substrate conditions are shown in Table 1 below.

Table 1 Broad-spectrum degradation of Acinetobacter baumannii DD1 substrates

substance boiling point Dosing concentration (mg/L) Degradation time Degradation rate phenol 181.9°C 94.11 20h 99.9% toluene 110.6°C 92.14 24h 50% Butanol 117.25°C 74.12 24h 99.9% Dioxane 101.3°C 88.11 40h 99.9% Tetrahydrofuran 66°C 72.11 70h 99.9% n-Hexane 68.74°C 86.17 108h 99.9% ethanol 78.5°C 46.07 24h 99.9% benzene 80.1°C 78 24h 99.9% O-xylene 140°C 68.8 60h 40%

As can be seen from the results of Table 1, Acinetobacter baumannii DD1 phenol of the present invention, n-butanol, dioxane, tetrahydrofuran, n-hexane, ethanol and benzene all have good degradation effect, and the degradation rate is average under its respective optimal culture time. It can reach 99.9%.

Claims (7)

1. An Acinetobacter baumannii, characterized in that it is named Acinetobacter baumannii DD1 (Acinetobacter baumannii DD1), and the preservation number is CCTCC NO: M 2013560. 2. an application of Acinetobacter baumannii as claimed in claim 1 in degrading dioxane. 3. the application of Acinetobacter baumannii as claimed in claim 2 in degrading dioxane, it is characterized in that, said Acinetobacter baumannii is inoculated to waste water containing dioxane, inorganic salt containing dioxane The culture medium or the inorganic salt medium with dioxane waste gas are cultivated to degrade dioxane. 4. The application of Acinetobacter baumannii in degrading dioxane according to claim 3, characterized in that the cultivation is carried out at a pH value of 5.0-8.0 and a temperature of 25°C-40°C. 5. the application of Acinetobacter baumannii in degrading dioxane as claimed in claim 3, is characterized in that, the initial concentration of dioxane in described waste water is 100mg/L～500mg/L; The initial concentration of dioxane in the inorganic salt culture medium of alkane is 100mg/L～500mg/L; The initial concentration of dioxane in the inorganic salt medium that is passed into the dioxane waste gas is 100mg/L～500mg/L L. 6. The application of Acinetobacter baumannii in degrading dioxane as claimed in claim 3, wherein the inorganic salt medium comprises the following substances: 3.5g Na ₂ HPO ₄ ·2H ₂ O, 1.0gKH ₂ Dissolve PO ₄ , 0.5g(NH ₄ ) ₂ SO ₄ , 0.1g MgCl ₂ ·6H ₂ O, 0.05g Ca(NO ₃ ) ₂ in 1000mL water, add 1ml trace element solution, and adjust the pH to 7.0-7.2; The composition of the trace element solution is: FeSO ₄ 7H ₂ O 1.0g, CuSO ₄ 5H ₂ O 0.02g, H ₃ BO ₃ 0.014g, MnSO ₄ 4H ₂ O 0.10g, ZnSO ₄ 7H ₂ O 0.10g , Na ₂ MoO ₄ ·2H ₂ O 0.02g, CoCl ₂ ·6H ₂ O 0.02g, dissolved in 1000mL of water. 7. An application of Acinetobacter baumannii as claimed in claim 1 in degrading phenol, toluene, n-butanol, n-hexane, ethanol, benzene, tetrahydrofuran or o-xylene.