CN103695351B - Acinetobacter baumannii and application thereof - Google Patents
Acinetobacter baumannii and application thereof Download PDFInfo
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Abstract
The invention discloses acinetobacter baumannii and application thereof. The acinetobacter baumannii is named as Acinetobacter baumannii DD1 which has the preservation number of CCTCC (China Center for Type Culture Collection) NO: M2013560 and is preserved in the CCTCC in Wuhan University of Wuhan in China on November 8th, 2013. The acinetobacter baumannii is inoculated into waste water containing dioxane or introduced into an inorganic salt culture medium of dioxane waste gas to be cultured. As a result, the dioxane can be completely degraded, and contaminants such as toluene, phenol and tetrahydrofuran can be degraded. The acinetobacter baumannii is a non-fermentation-type aerobiotic gram-negative dyeing bacterium which can grow by taking the dioxane as a sole carbon source and energy source and is capable of effectively degrading the dioxane substrate at the same time. The acinetobacter baumannii can be used for degrading various contaminants such as toluene, phenol and tetrahydrofuran. Thus, a foundation for engineering application of purifying wastewater or waste gas containing the dioxane via a biological method is laid.
Description
Technical field
The present invention relates to a kind of degradation method of diox, be specifically related to a kind of Acinetobacter baumannii (Acinetobacter baumannii) and application thereof.
Background technology
1,4-dioxane (1,4-dioxane), another name diox, having well water-soluble, is a kind of good organic solvent, can be compatible with ethanol, ether, acetone, phenol etc., be widely used in the industries such as paint, dyestuff, pharmacy, be also applied in the consuming product such as food, makeup and washing composition.Diox is irritant to skin, eye and respiratory system, can accumulate in human body, serious infringement is had to liver, kidney and neural system, chronic progressive poisoning can cause the disease such as uremia, renal failure, acute poisoning may cause death, is classified as B2 level (possible) human carcinogen by the international tumor research center (IARC) of Environmental Protection Agency (U.S.EPA) and the World Health Organization.
The extensive application of diox, causes the pollution of surface water and groundwater day by day serious.At present, in the underground water and landfill yard of the U.S., Canada and multiple developed country such as Japanese, the pollution of diox all detected; High density diox is detected in the underground water of the refuse landfill near Canadian Ottawa, Lake Ontario and Canada; China the Yellow River also detects a large amount of diox, and detects the accumulation of also Chu diox in the indigenous strains internal organ of Lanzhou section.More there are some researches show, even if also detect the existence of diox in hard to get to arctic frozen soil and underground water.Therefore, the removal of surface water and groundwater Zhong diox is very urgent, needs a kind of effective method badly and is removed.
The domestic and international method mainly chemical oxidization method for removing diox at present, comprise ozone, UV illumination, hydrogen peroxide and Fenton oxidation etc., these methods all have respective advantage, but this class methods processing cost is too high, is not suitable for the environmental pollution improvement of diox.There is due to diox the physico-chemical properties such as cyclic ether structure, C-O energy-rich bond, low Henry's constant and low octanol-water partition coefficient, be once classified as " not easily degradable chemical ".
1991, German scholar Bemhardt etc. isolated the bacterial strain Rhodococcus ruber219 with diox degradation capability first Cong the mud in diox chemical plant, and first time achieves biological degradation diox.Thus increasing investigator starts the research paying close attention to Biochemical method diox.1993, Burback etc. had and isolate the bacterial strain Mycobacterium vaccae that a strain has diox degradation capability, but this bacterial strain diox degradation capability is limited and can not continued propagation.1000mg/L diox can be degraded 90% by the mycobacterium PH-06 of the report such as Young-Mo Kim in 15 days, but degradation cycle is long.The isolated fungi Cordyceps such as Nakamiya sinensis also can using diox as sole carbon source and energy continued propagation.Current report diox degradation bacteria also has Pseudonocardia dioxanivorans CB1190, Pseudonocardia B5, Bacillus pumilus D4 and Xanthobacter D7 etc.
Summary of the invention
The invention provides a kind of Acinetobacter baumannii (Acinetobacter baumannii) and application thereof, (degradation rate of Acinetobacter baumannii) Dui diox reaches more than 99.9%, the pollutents such as toluene, phenol, tetrahydrofuran (THF) of can also degrading to this Acinetobacter baumannii.
A kind of Acinetobacter baumannii, called after Acinetobacter baumannii DD1(AcinetobacterbaumanniiDD1), preserving number is CCTCC NO:M2013560.
Acinetobacter baumannii DD1(Acinetobacter baumannii DD1 of the present invention) be preserved on November 8th, 2013 China typical culture collection center being positioned at Wuhan, China Wuhan University.
Acinetobacter baumannii DD1 of the present invention is collected in the second pond active sludge of sewage work, and shape is rod-short, and size is 1.0 ~ 1.5 × 1.5 ~ 2.5 μm, and atrichia, without gemma; After solid plate substratum 30 DEG C cultivates 48h, single bacterium colony is spherical shape, smooth moistening, and lawn is along line growth; Gramstaining is negative, and the experiment of oxydase, catalase is for negative; The experiment of gelatin, Citrate trianion, nitrate reduction is for positive; To kantlex, rifomycin, there is resistance, to tsiklomitsin non-resistant; Salinity is greater than more than 4% poor growth.
Present invention also offers a kind of as described in Acinetobacter baumannii degraded diox in application.
This application is specially: cultivate in the minimal medium described Acinetobacter baumannii being seeded to the waste water containing diox, minimal medium containing diox or passing into diox waste gas, degraded diox.
Described Acinetobacter baumannii DD1(Acinetobacter baumannii DD1) diox can be utilized as unique carbon source and energy substance growth and breeding, diox is mineralized into CO
2and H
2o.Under pure culture condition, this bacterium can be degradable by the 100mg/L diox in minimal medium in 42h.
Further preferably, described cultivation pH value be 5.0 ~ 8.0, temperature is carry out in the scope of 25 DEG C ~ 40 DEG C.Be more preferably pH value be 7.0, temperature is carry out in the scope of 32 DEG C.
Further preferably, the starting point concentration of described Fei water Zhong diox is 100mg/L ~ 500mg/L; The starting point concentration of the described minimal medium Zhong diox containing diox is 100mg/L ~ 500mg/L; The described starting point concentration passing into the minimal medium Zhong diox of diox waste gas is 100mg/L ~ 500mg/L.More preferably 200mg/L is.
The described starting point concentration passing into the minimal medium Zhong diox of diox waste gas is that 100mg/L ~ 500mg/L refers to: in minimal medium, diox starting point concentration refers to pass in diox waste gas and is dissolved in diox starting point concentration in inorganic salt culture medium, therefore the intake of ， diox waste gas is measured with starting point concentration needed for minimal medium Zhong diox.
Further preferably, incubation time is 20 ~ 50h, is more preferably 42h.
Further preferably, described minimal medium (MSM) comprises following material: 3.5gNa
2hPO
42H
2o, 1.0g KH
2pO
4, 0.5g (NH
4)
2sO
4, 0.1g MgCl
26H
2o, 0.05gCa (NO
3)
2, be dissolved in 1000mL water, add 1ml trace element solution, regulate pH to 7.0 ~ 7.2.The medium component part of the minimal medium containing diox or the minimal medium that passes into You diox waste gas is identical.
Described trace element solution consists of FeSO
47H
2o1.0g, CuSO
45H
2o0.02g, H
3bO
30.014g, MnSO
44H
2o0.10g, ZnSO
47H
2o0.10g, Na
2moO
42H
2o0.02g, CoCl
26H
2o0.02g, is dissolved in 1000mL water.
Acinetobacter baumannii of the present invention can also be used for degradation of phenol, toluene, tetrahydrofuran (THF) or o-Xylol.
Described Acinetobacter baumannii is seeded in the minimal medium containing phenol, toluene or tetrahydrofuran (THF) and cultivates, degradation of phenol, toluene or tetrahydrofuran (THF).Other degradation conditions are the same with degraded diox, and degradation time is different, and the degradation rate of bacterial strain Degradation of Phenol 20h of the present invention reaches 99.9%, reaches 50% to the degradation rate of degradation of toluene 24h, reach 99.9% to the degradation rate of tetrahydrofuran (THF) degraded 48h.
Acinetobacter baumannii of the present invention is also for propyl carbinol of degrading, normal hexane, ethanol or benzene, and degradation rate all reaches 99.9%.The method of degradation treatment is the same with degraded diox, and degradation time is different, and the best degradation time of propyl carbinol is 24h, and the best degradation time of normal hexane is 108h, and the best degradation time of ethanol is 24h, and the best degradation time of benzene is 24h.
Beneficial effect of the present invention is:
The invention provides the Acinetobacter baumannii of a strain energy efficient degradation diox, this bacterial strain is aerobic non-fermented type Gram-negative bacterium, and can be sole carbon source and energy growth this substrate of efficient degradation simultaneously with diox, degradation rate reaches more than 99%; This bacterial strain can be degraded the multiple pollutants such as toluene, phenol, tetrahydrofuran (THF); The present invention adopts the engineer applied that biological method purification contains diox waste water or waste gas to lay a good foundation.
Accompanying drawing explanation
Fig. 1 is Acinetobacter baumannii DD1 degraded diox and thalli growth situation;
Fig. 2 be different pH for cell concentration with the impact of diox degradation rate;
Fig. 3 is diox concentration changes with time situation Xia differing temps;
Fig. 4 is diox concentration changes with time situation Xia different diox starting point concentration;
Fig. 5 is Acinetobacter baumannii DD1 degradation of phenol and growth situation;
Fig. 6 is Acinetobacter baumannii DD1 tetrahydrofuran-degrading and growth situation.
Embodiment
Below in conjunction with specific embodiment, invention is described further, but protection scope of the present invention is not limited in this:
Minimal medium (MSM) comprises following material: 3.5g Na
2hPO
42H
2o, 1.0gKH
2pO
4, 0.5g (NH
4)
2sO
4, 0.1g MgCl
26H
2o, 0.05g Ca (NO
3)
2, be dissolved in 1000mL water, add 1ml trace element solution, regulate pH to 7.0 ~ 7.2.
Trace element solution consists of FeSO
47H
2o1.0g, CuSO
45H
2o0.02g, H
3bO
30.014g, MnSO
44H
2o0.10g, ZnSO
47H
2o0.10g, Na
2moO
42H
2o0.02g, CoCl
26H
2o0.02g, is dissolved in 1000mL water.
The Isolation and ldentification of embodiment 1:Acinetobacter baumannii DD1
(1) sample collecting and domestication
The active sludge of collection in worksite Hangzhou seven lattice secondary sedimentation tank of sewage treatment work take diox as sole carbon source and the energy, carries out taming, enrichment.After several months, the active sludge after enrichment, is inoculated in the 250mL triangular pyramidal bottle containing 100mL MSM substratum, using diox as sole carbon source and energy substance, continues cultivation, enrichment.Experiment needs constant temperature (30 ± 1 DEG C), and carries out under remaining on aerobic condition.
(2) isolation and identification for strains
Mixed bacteria liquid through enrichment of repeatedly going down to posterity is carried out gradient dilution, is inoculated on the inorganic salt nutrient agar only using diox as sole carbon source by the form of coating method, be placed in constant incubator (30 DEG C) and cultivate.Picking list bacterium colony.To single bacterium colony repeatedly rule be separated after, then take back the MSM substratum of Han diox 100mg/L) in cultivate, test its whether have degraded diox ability.Choose the bacterial classification with degradation capability and carry out further separation and purification, until isolate the bacterial strain DD1 with diox degradation capability.
Bacterial strain shape is rod-short, and size is 1.0 ~ 1.5 × 1.5 ~ 2.5 μm, and atrichia, without gemma; After solid plate substratum 30 DEG C cultivates 48h, single bacterium colony is spherical shape, faint yellow, smooth moistening, and lawn is along line growth; Gramstaining is negative; The experiment of gelatin, Citrate trianion, nitrate reduction is for positive; The experiment of oxydase, catalase is for negative; To kantlex, rifomycin, there is resistance, to tsiklomitsin non-resistant; Salinity is greater than more than 4% poor growth.
Above-mentioned feature matches with the Physiological-biochemical Characters of the acinetobacter that " the outstanding Bacteria Identification handbook of uncle " is edited and recorded.This bacterial strain is through Biolog microorganism identification and 16S rDNA homology analysis, in conjunction with the mycology feature of above Physiology and biochemistry, be accredited as Acinetobacter baumannii, the complete genome sequence of 16S rDNA is as shown in SEQ ID NO:1, and GenBank accession number is KF713537.
By this Acinetobacter baumannii called after Acinetobacter baumannii DD1(Acinetobacter baumanniiDD1), be deposited on November 8th, 2013 China typical culture collection center being positioned at Wuhan, China Wuhan University, preserving number is CCTCC NO:M2013560.
The characteristic of embodiment 2:Acinetobacter baumannii DD1 degraded diox
(1) diox, as the sole carbon source of Acinetobacter baumannii DD1, inoculates Acinetobacter baumannii DD1 thalline in 100mL minimal medium, initial thalline OD
600be 0.01; Adding diox makes initial diox concentration be 100mg/L.Being placed in temperature is 30 DEG C, and revolution is cultivate in the shaking table of 130r/min, regularly samples, and when cultivating 42h, the OD value of bacterial strain reaches maximum value 0.183, the results are shown in Figure 1.
As shown in Figure 1, along with the prolongation of time, the concentration of bacterial classification also constantly increases.The present embodiment illustrates that degradation bacteria Acinetobacter baumanniiDD1 can utilize diox to carry out biological self reproducing as sole carbon source and the energy, and has the ability of efficient degradation diox.
(2) regulate minimal medium to different pH value (5.0,6.0,6.5,7.0,7.5,8.0) with NaOH or HCl solution, under initial diox concentration is the condition of 100mg/L, access bacterial strain makes initial thalline OD
600be 0.01.Sample is placed in 30 DEG C, shaking culture in 130r/min constant-temperature table, sample after cultivating 35h, the results are shown in Figure 2.
As seen from Figure 2, in the scope of pH5.0 ~ 8.0, microorganism all can be degraded diox preferably, and with the growth of cell concn; Along with the continuous increase of pH, bacterial strain concentration and diox degraded take the lead in raising rear decline, and the optimal pH of degraded is 7.0.
(3) be in the minimal medium of 100mg/L in initial diox concentration, access bacterial strain makes initial thalline OD
600be 0.01.The temperature of each sample is set to respectively 25 DEG C, 30 DEG C, 32 DEG C, 37 DEG C, 40 DEG C, is placed in shaking culture in 130r/min constant-temperature table, timing sampling, measure the residual concentration of diox, the results are shown in Figure 3.As shown in Figure 3, in the scope of 25 DEG C ~ 40 DEG C, bacterial strain all can grow, and degradation rate has the process declined afterwards that first rises along with the rising of temperature, and 32 DEG C time, degradation rate reaches maximum.
(4) diox starting point concentrations are respectively in the minimal medium of 100mg/L, 200mg/L, 300mg/L, 400mg/L, 500mg/L, and access bacterial strain makes initial thalline OD
600be 0.01, sample be placed in 30 DEG C, shaking culture in 130r/min constant-temperature table, regularly sample, the results are shown in Figure 4.As shown in Figure 4, except 500mg/L, can be degradable, degradation rate has the process declined afterwards that first rises along with the rising of concentration, and wherein when 200mg/L, degradation rate is the fastest.
Embodiment 3:Acinetobacter baumannii DD1 degradation of phenol and tetrahydrofuran (THF)
Respectively using phenol and tetrahydrofuran (THF) as the sole carbon source of Acinetobacter baumannii DD1 and the energy, be all in the minimal medium of 100mg/L at starting point concentration, access bacterial strain makes initial thalline OD
600be 0.01, sample be placed in 30 DEG C, shaking culture in 130r/min constant-temperature table, regularly sample, the results are shown in Figure 5, Fig. 6.
From the result of Fig. 5, along with the prolongation of incubation time, the concentration of bacterial classification increases, and the concentration of phenol reduces, and when incubation time is more than 20h, phenol is degraded completely substantially;
From the result of Fig. 6, along with the prolongation of incubation time, the concentration of bacterial classification increases, and the concentration of tetrahydrofuran (THF) reduces, and when incubation time reaches 70h, tetrahydrofuran (THF) is degraded completely substantially;
Embodiment 4:Acinetobacter baumannii DD1 substrate broad spectrum degraded situation
In minimal medium, add various substrate (namely be respectively single carbon source with the substrate in table 1) respectively, dosage sees the following form 1, and access bacterial strain makes initial thalline OD
600be 0.01, sample is placed in 30 DEG C, shaking culture in 130r/min constant-temperature table, period sampling measuring, result shows, bacterial strain cannot directly utilize the pollutents such as trichloromethane, trieline, trichoroacetic acid(TCA), octane-iso, and the substrate situation that can directly utilize sees the following form 1.
Table 1Acinetobacter baumannii DD1 substrate broad spectrum degraded situation
| Material | Boiling point | Add concentration (mg/L) | Degradation time | Degradation rate |
| Phenol | 181.9℃ | 94.11 | 20h | 99.9% |
| Toluene | 110.6℃ | 92.14 | 24h | 50% |
| Propyl carbinol | 117.25℃ | 74.12 | 24h | 99.9% |
| Diox | 101.3℃ | 88.11 | 40h | 99.9% |
| Tetrahydrofuran (THF) | 66℃ | 72.11 | 70h | 99.9% |
| Normal hexane | 68.74℃ | 86.17 | 108h | 99.9% |
| Ethanol | 78.5℃ | 46.07 | 24h | 99.9% |
| Benzene | 80.1℃ | 78 | 24h | 99.9% |
| O-Xylol | 140℃ | 68.8 | 60h | 40% |
As shown in Table 1, Acinetobacter baumannii DD1 phenol of the present invention, propyl carbinol, diox, tetrahydrofuran (THF), normal hexane, ethanol and benzene all have good degradation effect, and under its respective best incubation time, degradation rate all can reach 99.9%.
Claims (7)
1. an Acinetobacter baumannii, is characterized in that, called after Acinetobacter baumannii DD1 (Acinetobacter baumannii DD1), preserving number is CCTCC NO:M 2013560.
2. the application of Acinetobacter baumannii in degraded diox as claimed in claim 1.
3. the application of Acinetobacter baumannii in degraded diox as claimed in claim 2, it is characterized in that, cultivate in the minimal medium described Acinetobacter baumannii being seeded to the waste water containing diox, minimal medium containing diox or passing into diox waste gas, degraded diox.
4. the application of Acinetobacter baumannii in degraded diox as claimed in claim 3, is characterized in that, described cultivation pH value be 5.0 ~ 8.0, temperature is carry out in the scope of 25 DEG C ~ 40 DEG C.
5. the application of Acinetobacter baumannii in degraded diox as claimed in claim 3, it is characterized in that, the starting point concentration of described Fei water Zhong diox is 100mg/L ~ 500mg/L; The starting point concentration of the described minimal medium Zhong diox containing diox is 100mg/L ~ 500mg/L; The described starting point concentration passing into the minimal medium Zhong diox of diox waste gas is 100mg/L ~ 500mg/L.
6. the application of Acinetobacter baumannii in degraded diox as claimed in claim 3, it is characterized in that, described minimal medium comprises following material: 3.5g Na
2hPO
42H
2o, 1.0gKH
2pO
4, 0.5g (NH
4)
2sO
4, 0.1g MgCl
26H
2o, 0.05g Ca (NO
3)
2, be dissolved in 1000mL water, add 1ml trace element solution, regulate pH to 7.0 ~ 7.2;
Described trace element solution consists of: FeSO
47H
2o 1.0g, CuSO
45H
2o 0.02g, H
3bO
30.014g, MnSO
44H
2o 0.10g, ZnSO
47H
2o 0.10g, Na
2moO
42H
2o0.02g, CoCl
26H
2o 0.02g, is dissolved in 1000mL water.
7. the application of Acinetobacter baumannii in degradation of phenol, toluene, propyl carbinol, normal hexane, ethanol, benzene, tetrahydrofuran (THF) or o-Xylol as claimed in claim 1.
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| CN104528949B (en) * | 2014-12-28 | 2016-05-11 | 国家电网公司 | A kind of technique of processing power plant Waste Water Containing Sulfur |
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| JP6835311B2 (en) * | 2016-12-08 | 2021-02-24 | 大成建設株式会社 | Biodegradation treatment method for cyclic ether |
| CN109628337B (en) * | 2018-11-20 | 2022-04-19 | 江苏宜裕环保科技有限公司 | Efficient phenol degrading bacterium and application thereof |
| CN111944719B (en) * | 2020-08-14 | 2022-04-29 | 暨南大学 | Dioxane degrading bacterium IS20 as well as preparation method and application thereof |
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| CN113186130B (en) * | 2021-04-26 | 2022-11-08 | 广东工业大学 | Acinetobacter baumannii GDUTAN8 and application thereof in degrading benzene series |
| CN115247140B (en) * | 2022-07-05 | 2023-04-07 | 湖北省生态环境科学研究院(省生态环境工程评估中心) | Bacterial strain for degrading petroleum hydrocarbon under arsenic stress and application thereof |
| CN116445368B (en) * | 2023-06-06 | 2023-09-19 | 碧沃丰生物科技(广东)股份有限公司 | Acinetobacter baumannii and application thereof |
| CN117343871B (en) * | 2023-09-28 | 2024-04-09 | 四川轻化工大学 | Acinetobacter baumannii, esterified liquid and application thereof in yellow water treatment |
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Effective date of registration: 20201222 Address after: No.477 Yongle Road, Wangdian Town, Xiuzhou District, Jiaxing City, Zhejiang Province Patentee after: ZHEJIANG OUTUO ELECTRICAL Co.,Ltd. Address before: 310018, No. 18 Jiao Tong Street, Xiasha Higher Education Park, Hangzhou, Zhejiang Patentee before: ZHEJIANG GONGSHANG University |