CN104263686A - Acinetobacter strain for low-temperature denitriding and application thereof - Google Patents
Acinetobacter strain for low-temperature denitriding and application thereof Download PDFInfo
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- CN104263686A CN104263686A CN201410513118.4A CN201410513118A CN104263686A CN 104263686 A CN104263686 A CN 104263686A CN 201410513118 A CN201410513118 A CN 201410513118A CN 104263686 A CN104263686 A CN 104263686A
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
Abstract
The invention discloses an acinetobacter strain for low-temperature denitriding and an application thereof and relates to an acinetobacter strain and an application thereof. The acinetobacter strain is preserved in China General Microbiological Culture Collection Center on September 1, 2014 and has the preservation number of CGMCC No. 9627. Since the acinetobacter strain bd-01 disclosed by the invention is a heterotrophic nitrification bacterium and can have a heterotrophic nitrification reaction at a low temperature of 6 DEG C so as to convert ammoniacal nitrogen into nitric nitrogen, nitrite nitrogen and intracellular substances. By adopting the acinetobacter strain, the total ammonia can be efficiently and thoroughly removed and the degradation rate of nitrogen is100% at the low temperature of 6 DEG C within 48 hours. The acinetobacter strain for low-temperature denitriding can be used for denitriding domestic sewage, agricultural wastes and industrial wastewater in areas with a low-emperature environment.
Description
Technical field
The present invention relates to strain acinetobacter calcoaceticus and an application thereof.
Background technology
Nitrogen content is the important indicator of sewage quality.In recent years, along with the fast development of China's economy, city domestic sewage quantity discharged is increasing, nitrogen content is more and more higher, and the nitrogen of high density can cause receiving water body oxygen deficit, grows harmful aquatic organisms, great loss is brought to agriculture production and aquaculture, more and more causes the concern of people.The denitrogenation processing of waste water has biological process and physico-chemical processes.But because the cost of physico-chemical process is higher, and easily cause secondary pollution, therefore its popularization is restricted.Biological denitrificaion method has the features such as economy, effective, easy to operate, non-secondary pollution, is acknowledged as the method with development prospect.
At present, biological denitrificaion method comprises traditional biological denitrificaion method and novel biological denitrificaion method.Traditional biological denitrificaion approach comprises aerobic nitrification and two stages of anaerobic denitrifying, is completed respectively by nitrifier and denitrifying bacteria.Because the flora participated in is different with Process operating parameters, nitrification and denitrification two process needs carry out in the reactor of two isolation, or the time or spatially cause replace anoxic and aerobic environment same reactor in carry out.
Discovered in recent years some allotrophic nitrobacterias, aerobic denitrifying bacteria and heterotrophic nitrification aerobic denitrifying bacteria, can complete denitrogenation whole process in same reaction vessel.So compared with traditional technology, the process advantage of SND is obvious, saves reaction vessel volume, shortens the reaction times, convenient operation and management, thus becomes one of focus of current biological denitrificaion research gradually.
But biological denitrificaion efficiency is by the restriction of temperature, and general bacterial strain is more weak at low temperature (<10 DEG C) denitrification ability.Especially at vast northern area, water temperature maintains 8-15 DEG C even lower throughout the year, and the microbic activity in biological treatment system receives serious suppression, and few about the research of Low temperature ammonia nitrogen at present.Therefore, carry out biological denitrificaion under cryogenic and become a great problem that northern area sewage treatment plants realizes qualified discharge.
This technology of biological denitrificaion has a extensive future, and has good social benefit, believes and can be widely used in the process of nitric wastewater from now on.But hanker after at present both at home and abroad the research such as clone of the separation to denitrogenation degradation bacteria strains, qualification, cultivation and correlation function gene more, and have no report about the safety research of denitrogenation bacterial strain, and the security of Thermal degradation bacterial strain is the important factor before bacterial strain application.It is that resident drinks qualified water, avoids the pathophorous generation of water, the important leverage of health of people.
Summary of the invention
The invention provides a strain acinetobacter calcoaceticus (Acinetobacter sp.), there is efficient cryogenic denitrification ability, the ammonia-state nitrogen of extensive concentration range of can degrading, the improvement of water body under low temperature environment and reparation are had great importance.
Low-temperature denitrification acinetobacter calcoaceticus of the present invention is acinetobacter calcoaceticus (Acinetobacter sp.) bd-01, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on September 1st, 2014, and deposit number is CGMCC No.9627.
Acinetobacter calcoaceticus bd-01 of the present invention is gram negative bacillus, aerobic, and without gemma, cell size is 1.5 μm × 2.0 μm; Bacterium colony is circular, smooth surface, neat in edge, and on LB substratum, bacterium colony is creamy white yellowish, cultivates in white clear in minimum medium.
Acinetobacter calcoaceticus bd-01 of the present invention, oxidase negative, catalase is positive, growth temperature range 6 ~ 30 DEG C, optimum growth temperature 15 ~ 25 DEG C; Growth pH scope 4.0 ~ 10.0, optimal pH is 7.0 ~ 9.0; Growth C/N=1 ~ 20, the suitableeest C/N=4 ~ 10.
Acinetobacter calcoaceticus bd-01 of the present invention can utilize organic and inorganic carbon nitrogenous source growth under 6 DEG C of conditions.This bacterial strain can grow in LB substratum and NYD substratum.Carbon source can utilize citric acid, Citrate trianion, glucose, sodium carbonate, sodium-acetate, glycerine and sucrose; Nitrogenous source can utilize the ammonium salts such as ammonium sulfate, ammonium chloride, ammonium molybdate, ammonium citrate.
Acinetobacter calcoaceticus bd-01 of the present invention passes through 16S rDNA sequence alignment analysis, with the 16S rDNA sequence homology 99% of the acinetobacter lwoffii (Acinetobacter lwoffii) of acinetobacter (Acinetobacter sp.).By belonging to acinetobacter (Acinetobacter sp.) in conjunction with morphological features, growth conditions, Physiology and biochemistry qualification result determination acinetobacter calcoaceticus bd-01, be acinetobacter lwoffii (Acinetobacter lwoffii).
Acinetobacter calcoaceticus bd-01 of the present invention can the NH of low middle and high concentration under 6 DEG C of conditions in effectively hydrolyzing water
4 +-N, 24 hours NH to low middle and high concentration
4 +-N (0.36 ~ 4.29mg/L, 48.32 ~ 159.98mg/L, 489.98 ~ 951.09mg/L) degradation rate is respectively 66.68 ~ 70.24%, 74.15% ~ 52.72%, 32.08% ~ 19.20%; 48 hours NH to low middle and high concentration
4 +the degradation rate of-N is respectively 70.70% ~ 72.91%, 82.43% ~ 92.82%, 44.98% ~ 27.99%.
Acinetobacter calcoaceticus bd-01 of the present invention is nitrification bacteria, can carry out heterotrophic nitrification reaction, ammonia-state nitrogen is converted into nitric nitrogen, nitrite nitrogen and intracellular matter under low temperature 6 DEG C of conditions.And the suitable growth conditions of this bacterial strain is wide in range, temperature 6 ~ 30 DEG C, C/N all can well-grown can remove total ammonia efficiently, up hill and dale in 7 ~ 9 scopes at 2 ~ 20, pH, is 100% under low temperature 6 DEG C of conditions in 48h to the degradation rate of nitrogen.
Acinetobacter calcoaceticus bd-01 of the present invention is the biological bacterial strain of one-level nontoxicity, mouse per os LD
50>15000mg/kg (approximately quite the lethal dose >1050g of body weight 70kg people).
Acinetobacter calcoaceticus of the present invention (Acinetobacter sp.) bd-01, belong to acinetobacter (Acinetobacter sp.), be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on September 1st, 2014, and deposit number is CGMCC No.9627.
Accompanying drawing explanation
Fig. 1 is the bacterium colony photo of acinetobacter calcoaceticus bd-01 of the present invention on LB substratum; Fig. 2 is the photo that acinetobacter calcoaceticus bd-01 of the present invention grows at minimal media liquid; The 16SrDNA sequence that Fig. 3 is the close bacterial strain in acinetobacter calcoaceticus bd-01 and GenBank of the present invention carries out the systematic evolution tree constructed by sequence analysis.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: present embodiment low-temperature denitrification acinetobacter calcoaceticus is acinetobacter calcoaceticus (Acinetobacter sp.) bd-01, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on September 1st, 2014, and deposit number is CGMCC No.9627.
Present embodiment acinetobacter calcoaceticus bd-01 is obtained by separation screening in the Songhuajiang River Water in Heilongjiang Province's March.Separation method carries out according to the following steps: get 100mL river and put into 500mL triangular flask, add 50mg ammonium sulfate, 300mg sodium-acetate and 5mL trace element solution, in 6 DEG C, 160r/min enrichment culture 5 days, getting 1mL enrichment culture liquid adds in the triangular flask containing 50mL minimum medium, 3 Duplicate Samples, in 6 DEG C, 160r/min shakes cultivation 3 days, by nutrient solution doubling dilution to 1000 times, getting 100 μ L nutrient solutions is coated on the solid plate of minimum medium, flat board is placed in 6 DEG C of incubators and cultivates until grow single bacterium colony, the test of low temperature ammonia nitrogen degradation is carried out by after single bacterium colony line purifying, Low temperature ammonia nitrogen strain can be obtained---acinetobacter calcoaceticus bd-01.
Minimum medium formula (1000mL): be made up of 0.5g/L ammonium sulfate, 5g/L sodium-acetate, 50mL trace element solution and 950mL water, 121 DEG C of sterilizing 20min.Described trace element solution (g/L): be made up of dipotassium hydrogen phosphate 5g, magnesium sulfate 2.5g, sodium-chlor 2.5g, ferric sulfate 0.05g, manganous sulfate 0.05g and 1L water.
Carry out the qualification of form, cultural characters and physio-biochemical characteristics to bacterial strain, concrete steps are as follows: under 1000 power microscopes, observe thalline size and thalline color after the Low-temperature culture thalline of 48 hours is carried out gramstaining; To be coated on minimum medium and organism substratum (NYD) flat board after bacterium liquid doubling dilution, observe colonial morphology and color; Liquid bacteria liquid is cultivated 4-7 days, observes and have non-pigment to produce; Bacterial strain is carried out oxydase, catalase, arginine dihydrolase, dextrose plus saccharose ferments, V.P, M.R and gelatin liquification test.This bacterial strain of result is gram negative bacillus, aerobic, and without gemma, cell size is 1.5 μm × 2.0 μm; Bacterium colony is circular, smooth surface, neat in edge, and on LB substratum, bacterium colony is creamy white yellowish (as Fig. 1), cultivates in white clear (as Fig. 2) in minimum medium.Oxidase negative, catalase is positive, and arginine dihydrolase is positive, utilizes dextrose plus saccharose, and V.P is negative, and M.R is negative, gelatine liquefication, growth temperature range 6-30 DEG C, optimum growth temperature 15-25 DEG C.
16S rDNA sequential analysis is carried out to bacterial strain: get 1mL bacterium liquid collected by centrifugation thalline, add 100 μ L sterilized waters, 10min is boiled after suspension, collected after centrifugation supernatant liquor obtains STb gene, pcr amplification is carried out with bacterial 16 S rDNA universal primer, PCR primer is carried out agarose gel electrophoresis, reclaim size to check order at the unique DNA band at 1500bp place, result obtains 1388bp DNA sequence dna, homologous sequence comparison is carried out in GenBank, with Mega 4.0 software building phylogenetic tree (as Fig. 3), the 16S rDNA sequence homology 99% of the acinetobacter lwoffii (Acinetobacter lwoffii) of result and acinetobacter (Acinetobacter sp.), combining form, cultural characters and physio-biochemical characteristics, be accredited as acinetobacter lwoffii (Acinetobacter lwoffii).
Embodiment two: present embodiment acinetobacter calcoaceticus (Acinetobacter sp.) bd-01 is used for the denitrogenation of low temperature environment area sanitary sewage, agricultural wastes and trade effluent, and described low temperature is 6 DEG C.
Present embodiment acinetobacter calcoaceticus bd-01 has low temperature (6 DEG C) efficient denitrification ability, the ammonia-state nitrogen (0.50mg/L-951.09mg/L) of extensive concentration range of can degrading, can be applicable to the denitrogenation of the low temperature environment area water bodys such as sanitary sewage, agricultural wastes and trade effluent, the improvement of water body under low temperature environment and reparation are had great importance.
For verifying the effect of acinetobacter calcoaceticus bd-01 of the present invention, carry out following experiment:
One, the culture condition of low-temperature denitrification acinetobacter calcoaceticus.Be inoculated in minimum medium by bacterium liquid with the identical bacterium amount that connects, respectively at 6 DEG C, 8 DEG C, 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 160r/min shakes cultivation 2 days, with spectrophotometric determination bacterium turbidity (OD
600).The results are shown in Table 1,6-30 DEG C of thalline all can grow, 15 DEG C, 20 DEG C and 25 DEG C of thalli growths the fastest, 6 DEG C, 8 DEG C, 10 DEG C and 30 DEG C of thalli growths very fast, in 48h, the degradation rate of nitrogen all reaches 100%.
Table 1 bacterial strain growing state at different temperatures
By initial incubation liquid sodium hydroxide and hydrochloric acid respectively furnishing pH be 4,5,6,7,8,9,10, by identical inoculum size inoculation bacterium liquid, 6 DEG C, 160r/min shakes cultivation 2 days, measures bacterium turbidity OD
600value.The results are shown in Table 2, pH very fast at 7-9 thalli growth, the nitrogen degradation rate of 24 hours just all reached more than 85%, pH4,5,6,10 thalline allometrys are comparatively slow, the degradation rate of nitrogen is lower.
The growing state of table 2 bacterial strain under different PH
The pH value of fixing nutrient solution is 7.2, and adjustment C/N is 1-20, identical inoculum size, 6 DEG C, and 160r/min shakes cultivation 2 days, measures bacterium turbidity OD
600value.The results are shown in Table 3, C/N all can grow at 1-20 thalline, C/N is very fast at 2-20 thalli growth.24 hours C/N be 6, the degradation rate of bacterium to nitrogen that grow under 9-15 condition be all greater than 96.8%; The bacterial strain grown under C/N is 4-20 condition for 48 hours to the degradation rate of nitrogen all close to 100%.
Table 3C/N is on the impact of strain growth
| C/N | Initial OD 600 | OD600(24h) | OD(48h) |
| 1 | 0.011±0.12 | 0.166±0.22 | 0.186±0.23 |
| 2 | 0.013±0.14 | 0.421±0.17 | 0.498±0.28 |
| 3 | 0.022±0.16 | 0.496±0.11 | 0.520±0.25 |
| 4 | 0.013±0.12 | 0.629±0.16 | 0.698±0.18 |
| 5 | 0.011±0.17 | 0.538±0.14 | 0.628±0.27 |
| 6 | 0.015±0.13 | 0.706±0.12 | 0.707±0.22 |
| 7 | 0.013±0.14 | 0.615±0.11 | 0.625±0.24 |
| 8 | 0.009±0.17 | 0.536±0.15 | 0.642±0.27 |
| 9 | 0.013±0.22 | 0.631±0.17 | 0.646±0.24 |
| 10 | 0.010±0.16 | 0.636±0.21 | 0.644±0.21 |
| 12 | 0.017±0.12 | 0.698±0.22 | 0.699±0.23 |
| 15 | 0.015±0.11 | 0.647±0.16 | 0.666±0.26 |
| 20 | 0.013±0.12 | 0.685±0.18 | 0.690±0.19 |
Two, the heterotrophic nitrification of low-temperature denitrification acinetobacter calcoaceticus.
Bacterial strain is to the utilization of different carbon source: take ammonium sulfate as only nitrogen source, and change carbon source, carbon source is respectively citric acid, glucose, sodium carbonate, glycerine, ethanol, sodium-acetate and sucrose.Ammonium sulphate content in nutrient solution is 0.05%, and carbon source content is 0.5%, trace element 5%, pH7.2.50mL nutrient solution is contained in 250mL triangular flask, 6 DEG C, and 160r/min shakes cultivation 2 days, each process 3 Duplicate Samples, measures bacterium turbidity (OD
600).The results are shown in Table 4, bacterial strain, can comparatively good utilisation to all the other 6 kinds of carbon sources except can not grow in ethanol, and the utilizing status of Dichlorodiphenyl Acetate salt is best.
Table 4 different carbon source is on the impact of strain growth
Bacterial strain is to the utilization of different nitrogen sources: take sodium-acetate as sole carbon source, and change nitrogenous source, nitrogenous source is respectively ammonium sulfate, ammonium chloride, ammonium molybdate, ammonium citrate, saltpetre and Sodium Nitrite.Sodium acetate content in nutrient solution is 0.5%, and nitrogenous source content is 0.05%, trace element 5%, pH7.2.50mL nutrient solution is contained in 250mL triangular flask, 6 DEG C, and 160r/min shakes cultivation 2 days, each process 3 Duplicate Samples, measures bacterium turbidity (OD
600).The results are shown in Table 5, except saltpetre and Sodium Nitrite two kinds of nitrogenous sources, bacterial strain well can utilize all the other four kinds of nitrogenous sources, wherein best to the utilizing status of ammonium citrate.
Table 5 different nitrogen sources is on the impact of strain growth
Bacterial strain is to the degradation capability of the nitrogenous source of different concns: take sodium-acetate as sole carbon source, and nitrogenous source is ammonium sulfate, and concentration is divided into 3 sections, 0.50-5mg/L, 50-200mg/L, 500-1000mg/L.50mL nutrient solution is contained in 250mL triangular flask, 6 DEG C, and 160r/min shakes cultivation 2 days, and each process 3 is parallel, measures bacterium turbidity (OD
600).The results are shown in Table 6,24h bacterial strain to starting point concentration is that the degradation rate of nitrogen in 50mg/L nutrient solution is the highest; 48h bacterial strain is that the degradation rate of nitrogen in the nutrient solution of 100mg/L is the highest to starting point concentration, the degradation rate of starting point concentration to be 100-200mg/L higher than starting point concentration be all nitrogen in 50mg/L nutrient solution.Wherein, bacterial strain to the degradation capability of high density nitrogenous source (>500mg/L) lower than low middle concentration.
Table 6 bacterial strain is to the degradation effect of different concns nitrogenous source
Three, the studies on acute toxicity of low-temperature denitrification acinetobacter calcoaceticus
Test is divided into experimental group and blank group at random with mouse, often organizes 10, male and female half and half, body weight 18-22g.Overnight fast before administration, after claiming quality on an empty stomach, experimental mice is by a maximum dosage-feeding method, and per os gavage, dosage is 15000mg/kg, and blank group fills with the physiological saline of same volume, observes one week.Result experimental mice hair color, skin, mucous membrane, eyes, respiration cycle, autonomic activities and central nervous system behavior expression etc. all with blank group mouse without significant difference, occur without dead mouse in whole trial period.The LD of this bacterial strain
50>15000mg/kg (approximately quite the lethal dose of body weight 70kg people is 1050g), belongs to one-level non-toxic type material.
Claims (2)
1. a strain low-temperature denitrification acinetobacter calcoaceticus, it is characterized in that this bacterial strain is acinetobacter calcoaceticus (Acinetobacter sp.) bd-01, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on September 1st, 2014, and deposit number is CGMCC No.9627.
2. low-temperature denitrification acinetobacter calcoaceticus described in claim 1 is used for the denitrogenation of low temperature environment area sanitary sewage, agricultural wastes and trade effluent, and described low temperature is 6 DEG C.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434428A (en) * | 2016-08-30 | 2017-02-22 | 盐城师范学院 | Acinetobacter and application thereof |
| CN109082387A (en) * | 2018-03-14 | 2018-12-25 | 重庆理工大学 | It is a kind of can low temperature remove heterotrophic nitrification-aerobic denitrification composite bacteria agent and its application of high ammonia nitrogen |
| CN116286477A (en) * | 2023-01-16 | 2023-06-23 | 哈尔滨工业大学 | Low-temperature denitrification and dephosphorization deep blue-violet bacillus strain and application thereof |
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2014
- 2014-09-29 CN CN201410513118.4A patent/CN104263686B/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434428A (en) * | 2016-08-30 | 2017-02-22 | 盐城师范学院 | Acinetobacter and application thereof |
| CN109082387A (en) * | 2018-03-14 | 2018-12-25 | 重庆理工大学 | It is a kind of can low temperature remove heterotrophic nitrification-aerobic denitrification composite bacteria agent and its application of high ammonia nitrogen |
| CN116286477A (en) * | 2023-01-16 | 2023-06-23 | 哈尔滨工业大学 | Low-temperature denitrification and dephosphorization deep blue-violet bacillus strain and application thereof |
| CN116286477B (en) * | 2023-01-16 | 2023-12-05 | 哈尔滨工业大学 | Low-temperature denitrification and dephosphorization deep blue-violet bacillus strain and application thereof |
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