CN106591181B - A kind of Mysore arthrobacterium and its application in purifying sea water cultivation nitrogenous effluent - Google Patents
A kind of Mysore arthrobacterium and its application in purifying sea water cultivation nitrogenous effluent Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
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Abstract
The invention discloses a kind of Mysore arthrobacteriums, the bacterial strain preservation title is Mysore arthrobacterium 7-3, the bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and the deposit date is on October 17th, 2016, deposit number was CGMCC NO.13118.Meanwhile Mysore arthrobacterium provided by the invention can efficiently remove the ammonia nitrogen and nitrite in water body, can cultivate in nitrogenous effluent and be applied in purifying sea water.Mysore arthrobacterium detergent power provided by the invention is very strong, the purified treatment for being applied to nitrogenous effluent is not only easy to operate, can by waste water ammonia nitrogen and nitrite efficient removal fall, and it not will cause further pollution, it solves the problems, such as to handle in waste water in the prior art and needs special processing equipment, at high cost and poor processing effect, be suitable for being widely used in the purification of sea-farming nitrogenous effluent.
Description
Technical field
The present invention relates to microorganism and purposes, specifically a kind of Mysore arthrobacterium and its purifying sea water cultivation contain
Application in nitrogen waste water.
Background technique
In recent years, with the continuous development of culture fishery, sea-farming biological density improves year by year, in sea-farming
Excess in journey due to feed feeds, a large amount of discharges and the improper use of drug of aquatic livestock excreta, causes cultivation water
Body pollution is continuously increased, to generate murder by poisoning to aquatic livestock, is caused aquatic livestock morbidity even dead, has been seriously affected water
Produce the yield and benefit of cultivation.In general, the more serious pollutant of the seawater pollution of aquaculture is ammonia nitrogen and nitrous acid
Salt.Therefore, the ammonia nitrogen and nitrite pollution in breeding seawater how to be solved the problems, such as, it has also become current purge breeding seawater is ground
Study carefully hot and difficult issue.
Currently, being applied to the method for purifying sea water cultivation nitrogenous effluent mainly has physical method, chemical method and bioanalysis.Its
In, physical method and chemical method operation are relatively complicated, need special equipment, and operation operating cost is higher, and both
Method is low to organic removal rate in waste water, be easy to cause secondary pollution, therefore by very overall situation in actual popularization and application
Limit.Bioanalysis be efficiently use or convert the pollutants such as ammonia nitrogen and nitrite in water body by screening microorganism, thus
The ammonia nitrogen and nitrite concentration in water body are reduced, opposite physical method is compared with chemical method to be had safely and effectively, without secondary dirt
The advantages such as dye, at low cost, ecological, environmental protective.
Traditional biological denitrogenation processing includes two processes of nitrification and denitrification, and participating in biological denitrificaion microorganism mainly includes nitre
Change bacterium and denitrifying bacteria.Conventionally denitrification only occurs under anaerobism or anoxia condition, in the process,
Nitrate is successively reduced to nitrous acid, NO, N2O and N2.In recent years, with the discovery of heterotrophic nitrification aerobic denitrifying bacteria, it is
Biological denitrificaion provides a new way.Currently, both at home and abroad to the separation of aerobic denitrifying bacteria, screening and mechanism study compared with
It is more, and efficiently remove the dominant bacteria of nitrogen-containing pollutant and its in purifying aquaculture waste water application it is less, even if having
Several researchs report few in number, is also suitable only for the purification of the lower brackish water of salt content, because for universal microorganism
For growth, the salinity of growing environment is only one thousandth two, and the relatively high environment of seawater salinity can inhibit its growth, from
And it is not used to the purification higher seawater nitrogenous effluent of salt content.Therefore, it screens the fast-growth under hypersaline environment and there is height
The bacterial strain of nitrogen removal characteristics is imitated, and then is applied to the purified treatment of the higher sea-farming nitrogenous effluent of salt content, this is to sea
The protection of water breeding water body has a very important significance.
Summary of the invention
An object of the present invention is to provide a kind of Mysore arthrobacterium, nitrogenous to be applied to high salinity sea-farming
In the denitrogenation purified treatment of waste water.
It is existing to solve the second object of the present invention is to application of the Mysore arthrobacterium in purifying sea water cultivation nitrogenous effluent
With the presence of the problem of denitrogenation processing of the method to sea-farming nitrogenous effluent is at high cost, effect is poor, complex disposal process.
The purpose of the present invention is what is be achieved through the following technical solutions: a kind of Mysore arthrobacterium, the bacterial strain preservation name
Title is Mysore arthrobacterium (Arthrobacter mysorens) 7-3, which is preserved in Chinese microorganism strain preservation management
Committee's common micro-organisms center, the deposit date is on October 17th, 2016, deposit number was CGMCC NO.13118.
The cellular morphology of the bacterial strain of the present invention is in the shape of a rod, and bacterium colony is rounded, neat in edge rule, opaque,
Bacterium colony is in light yellow, colony diameter 0.5-1mm；For gram-positive bacterium, oxidizing ferment experiment is feminine gender, and catalase experiment is sun
Property, it is feminine gender that beta galactosidase, lysine decarboxylase and indoles, which generate experiment, and VP experiment is the positive.
Mysore arthrobacterium (Arthrobacter mysorens) 7-3 of the present invention uses bacterial 16 S rRNA gene
Universal primer carries out PCR amplification to the gene of sample, and amplification carries out sequencing through Institute of Microorganism, Academia Sinica,
The 16S rRNA gene order of the Mysore arthrobacterium is as shown in SEQ ID NO:1；Measurement result submits GenBank data
Library is through Blast sequence analysis, the results showed that the 16S rRNA of Mysore arthrobacterium (Arthrobacter mysorens) 7-3
Gene order and Arthrobacter mysorens similarity reach 99.23%.Through Institute of Microorganism, Academia Sinica according to
The experimental datas comprehensive analysis such as cellular morphology, physiological and biochemical property, the 16sRNA gene order of strain, with reference to " primary Jie Shi system
Bacteriology handbook " and International Journal of Systematic and Evolutionary
The related research paper of Microbiology, bacterial strain provided by the invention are Mysore arthrobacterium (Arthrobacter
mysorens)。
Mysore arthrobacterium (Arthrobacter mysorens) 7-3 provided by the invention be experimentally confirmed its 20 DEG C,
The salinity of 10-80 ‰ and it is aerobic under the conditions of, the ammonia nitrogen and nitrite in water body can be efficiently removed, it follows that it can be
It is applied in purifying sea water cultivation nitrogenous effluent.
The present invention also provides a kind of methods of purifying sea water cultivation nitrogenous effluent, by the entitled Mysore arthrobacterium of preservation
The activated seed liquid of (Arthrobacter mysorens) 7-3 is added in the sea-farming nitrogenous effluent that salinity is 30 ‰, in
PH value is 7.0-7.2, shaking table vibrates 48-72h under conditions of 20 DEG C；The oscillation rate of its shaking table oscillation is preferably that 150rpm is
Preferably.Its Mysore arthrobacterium (Arthrobacter mysorens) 7-3 is preserved in China Committee for Culture Collection of Microorganisms
Common micro-organisms center, the deposit date is on October 17th, 2016, deposit number was CGMCC NO.13118.
The method of purifying sea water cultivation nitrogenous effluent provided by the invention is by the Mysore arthrobacterium
(Arthrobacter mysorens) 7-3 is activated, and connects 3 ring of slant strains to three of the LB liquid medium equipped with 50mL
In the bottle of angle, 150r/min activates 12h, and the bacterium solution of activation is connected to the LB liquid medium equipped with 100mL by 5% inoculum concentration
In triangular flask, 150r/min shaking flask for 24 hours, obtains activated seed liquid, by Mysore arthrobacterium (Arthrobacter mysorens) 7-
3 activated seed liquid be added to salinity be 30 ‰ sea-farming nitrogenous effluent in, in pH value be 7.0-7.2,20 DEG C of condition
Lower shaking table oscillation degradation 48-72h, can be completed denitrogenation processing.Wherein Mysore arthrobacterium (Arthrobacter mysorens)
Active bacteria in the activated seed liquid of 7-3 is 108A CFU/mL, inoculum concentration 5% are 20 in preferred purified treatment temperature
DEG C, when preferably the pH value of environment purification is 7, it containing ammonia nitrogen or nitrite, salinity is 30 ‰ that bacterial strain, which is 100mg/L to concentration,
Waste water 48h removal efficiency up to 100%, to the compound nitrogen source containing ammonia nitrogen (100mg/L) and nitrite (50mg/L),
The removal efficiency of the 72h for the waste water that salinity is 30 ‰ is up to 92% or more.
Mysore arthrobacterium (Arthrobacter mysorens) 7-3 detergent power provided by the invention is very strong, is answered
Purified treatment for nitrogenous effluent is not only easy to operate, can by waste water ammonia nitrogen and nitrite efficient removal fall, and
And not will cause further pollution, can efficiently solve in the prior art handle waste water in need special processing equipment,
The problem of at high cost and poor processing effect is suitable for being widely used in the purification of high salinity sea-farming nitrogenous effluent.
The method for preserving of Mysore arthrobacterium (Arthrobacter mysorens) 7-3 provided by the invention: ultralow temperature freezes
Knot.
Detailed description of the invention
Fig. 1 is the Gram's staining for Mysore arthrobacterium (Arthrobacter mysorens) 7-3 that embodiment 1 is screened
Figure.
Fig. 2 is the growth curve chart for Mysore arthrobacterium (Arthrobacter mysorens) 7-3 that embodiment 1 is screened.
Specific embodiment
Following example is for present invention be described in more detail, but the invention is not limited in any way.
Embodiment 1: the screening of Mysore arthrobacterium (Arthrobacter mysorens) 7-3.
(1) packing 250mL enrichment culture is based on 500mL triangular flask, and several beades are added, and 25mL is added after sterilizing and adopts the Qin
Emperor island seawater-culture pond bed mud mud sample, at 30 DEG C, 100r/min shaken cultivation 2-3d；It draws culture solution 25mL and accesses new enrichment
In culture medium, at 30 DEG C, 100r/min shaken cultivation 2-3d；, operate 3 times repeatedly, purpose bacteria culture fluid must be enriched with；
Enriched medium: sodium citrate 5.0g/L, KNO32.0g/L, K2HPO41.0g/L, KH2PO41.0g/L
MgSO4·7H2O0.2g/L, NaCl 30.0g/L；
(2) it isolates and purifies: taking the above-mentioned enrichment purpose bacteria culture fluid of 10mL into 90mL sterile water, gradient dilution is coated on
BTB plate, 30 DEG C of culture 2-3d.There is the single colonie of blue halos from picking on BTB plate, continues on common bacteria plate point
From obtaining Mysore arthrobacterium (Arthrobacter mysorens) 7-3 pure culture.
Isolation medium: sodium citrate 5.0g/L, KNO32.0g/L, K2HPO41.0g/L, KH2PO41.0g/L
MgSO4·7H2O0.2g/L, NaCl 30.0g/L, 1%BTB ethanol solution 1mL, agar 20.0g/L.
(3) growth characteristics of bacterial strain: by the strain inoculated isolated and purified into beef extract-peptone slant medium, 20 DEG C
48-72h is cultivated, then from one ring of picking on beef extract-peptone slant medium into the 50mL triangular flask equipped with 30mL culture medium
12h is activated under same condition of culture, bacterium solution will have been activated and be inoculated into the beef extract egg equipped with 100mL by 5% inoculum concentration
In white peptone culture medium 250mL triangular flask, OD value is measured every 3h.The growth curve of the bacterial strain is as shown in Figure 2.
Researching and developing the bacterial strain that the discovery present invention screens in experimentation in inventor can under 10 ‰, 40 ‰ and 80 ‰ salinity
Enough growths, the most suitable growth salinity 30 ‰, but be below in 10 ‰ brackish water grow it is weak, so the bacterial strain is not suitable for nitrogenous
It is used in the purification of brackish water.
(4) identification of Mysore arthrobacterium (Arthrobacter mysorens) 7-3
A, strain morphology observation and Gram's staining identification:
Bacterium colony and bacterial strain observation: Mysore arthrobacterium (Arthrobacter mysorens) 7-3 is on LB solid medium
48h is cultivated, it is rounded to observe its bacterium colony, neat in edge rule, and opaque, bacterium colony is in light yellow, colony diameter 0.5-1mm.
B, Physiology and biochemistry detects:
Oxidizing ferment, contact enzymatic determination etc. have been carried out according to the related content that genus and species in " common bacteria identification handbook " are identified
Test.Bacterial strain uses Biolog measuring to the utilization of carbon source, and test result is as shown in table 1.The gram of the bacterial strain contaminates
Color result is as shown in Figure 1.
The Physiology and biochemistry testing result of 1 Mysore arthrobacterium of table (Arthrobacter mysorens) 7-3
C, molecular biology identification:
The obtained genomic DNA of Mysore arthrobacterium (Arthrobacter mysorens) B21-3 is detected, using drawing
Object carries out PCR amplification to bacterial strain DNA, and amplification submits GenBank database same through Blast through sequencing, measurement result
Source property compares, and reaches 99.23% with Arthrobacter mysorens similarity, and with reference to " primary Jie Shi Systems Bacterial learns to do
Volume " and International Journal of Systematic and Evolutionary Microbiology it is related
Research paper, the results showed that this bacterial strain are as follows: Mysore arthrobacterium Arthrobacter mysorens；The bacterial strain is preserved in China
Microbiological Culture Collection administration committee common micro-organisms center, the deposit date is on October 17th, 2016, deposit number was
CGMCC NO.13118。
Purification of embodiment 2 Mysore arthrobacterium (Arthrobacter mysorens) 7-3 to sea-farming nitrogenous effluent
Research
One, the preparation of Mysore arthrobacterium (Arthrobacter mysorens) 7-3 activated seed liquid:
The preparation of activated seed liquid: 3 ring of slant strains is connect into the triangular flask equipped with 50mL LB liquid medium, 150r/
Min activates 12h；The bacterium solution of activation is connected in the triangular flask of the LB liquid medium equipped with 100mL by 5% inoculum concentration,
At 20 DEG C, 150r/min is cultivated for 24 hours, and thalline were collected by centrifugation by 8000rpm, brine three times, resuspended bacterium solution, and guaranteeing outstanding
Active bacteria is 10 in bacterium solution8A CFU/mL；LB liquid medium (g/L): peptone 10.0, beef extract 3.0, NaCl 30.0, pH
7.0-7.2。
Two, degradation of Mysore arthrobacterium (Arthrobacter mysorens) 7-3 to ammonia nitrogen: activated seed liquid is pressed
It is inoculated into the culture medium containing heterotrophic nitrification containing 100mg/L according to 5% inoculum concentration, in 20 DEG C, 150r/min shaking table culture, often
Every ammonia nitrogen concentration in sample detection water body for 24 hours.
The preparation of shown heterotrophic nitrification culture medium: (NH4)2·SO4: 0.47g/L；Sodium citrate: 8.2g/L；NaCl
30‰；50mL Vickers salting liquid；PH:7.0-7.2；Wherein Vickers salting liquid: K2HPO4·3H2O:5.0g/L；FeSO4·7H2O:
0.05g/L；MgSO4·7H2O:2.5g/L；MnSO4·4H2O:0.05g/L.
The calculation formula of removal rate:
Removal rate=[c1- c2]/c1, wherein c1Initial concentration by adding nitrogen in system, c2To be remained in system after culture
The concentration of remaining added nitrogen.
Detecting water body, the results are shown in Table 2.
Removal efficiency of the 2 Mysore arthrobacterium of table to ammonia nitrogen
The result shows that ammonia nitrogen is removed completely when bacterial strain 48h provided by the invention.
Three, degradation of Mysore arthrobacterium (Arthrobacter mysorens) 7-3 to nitrite: by activated seed
Liquid is inoculated into the culture medium containing denitrification containing 50mg/L according to 5% inoculum concentration, in 20 DEG C, 150r/min shaking table culture,
Every sample detection Nitrite concentration for 24 hours.
The preparation of denitrification culture medium: NaNO2: 0.5g/L；KH2PO4: 1.0g/L；MgSO4·7H2O:0.2g/L；Citric acid
Sodium: 4.2g/L；FeSO4·7H2O:0.04g/L；MnSO4·4H2O:0.04g/L；NaCl 30‰；PH:7.0-7.5.
Detecting water body, the results are shown in Table 3.
3 Mysore arthrobacterium of table goes division result to nitrite nitrogen
The result shows that nitrite nitrogen is removed by Mysore Arthrobacter strain provided by the invention completely when 48h.
Four, Mysore arthrobacterium (Arthrobacter mysorens) 7-3 is to the composite nitrogen containing ammonia nitrogen and nitrite
Source degradation: activated seed liquid is inoculated into the ammonia nitrogen containing 100mg/L and the nitrite of 50mg/L according to 5% inoculum concentration
In complex medium, in 20 DEG C, 150r/min shaking table culture, every ammonia nitrogen, nitrite concentration in sample detection water body for 24 hours.
The preparation of complex medium: NaNO2: 0.25g/L；(NH4)2·SO4: 0.47g/L；KH2PO4: 1.0g/L；K2HPO4·3H2O:
1.0g/L；MgSO4·7H2O:0.2g/L；Sodium citrate: 12g/L；CaCl2: 0.1g/L；NaCl 30‰；PH:7.0-7.5.
Detecting water body, the results are shown in Table 4.
Removal efficiency of the 4 Mysore arthrobacterium of table to compound nitrogen source (ammonia nitrogen and nitrite nitrogen)
The result shows that: when 48h, Mysore Arthrobacter strain is 93.5% to ammonia nitrogen removal frank, to nitrite removal rate
It is 91.3%；It is 94.1% to ammonia nitrogen removal frank when 72h, is 92.1% to nitrite removal rate, shows to compound nitrogen source
Removing ability outstanding.
Above the present invention is described in detail with a general description of the specific embodiments, but in the present invention
On the basis of, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore, not
These modifications or improvements on the basis of deviation spirit of that invention, fall within the scope of the claimed invention.
SEQUENCE LISTING
<110>University Of Hebei
<120>a kind of Mysore arthrobacterium and its application in purifying aquatic product cultivating nitrogenous effluent
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1331
<212> DNA
<213>the 16s rRNA of Mysore arthrobacterium (Arthrobacter mysorens) 7-3
<400> 1
gcttcgggtg ttaccaactt tcgtgacttg acgggcggtg tgtacaaggc ccgggaacgt 60
attcaccgca gcgttgctga tctgcgatta ctagcgactc cgacttcatg gggtcgagtt 120
gcagacccca atccgaactg agaccggctt ttagggatta gctccacctc acagtatcgc 180
aacccattgt accggccatt gtagcatgcg tgaagcccaa gacataaggg gcatgatgat 240
ttgacgtcat ccccaccttc ctccgagttg accccggcag tctcccatga gtccccacca 300
ctacgtgctg gcaacatgga acgagggttg cgctcgttgc gggacttaac ccaacatctc 360
acgacacgag ctgacgacaa ccatgcacca cctgtgaacc agccccgaag ggaaacccca 420
tctctggagc ggtctggcac atgtcaagcc ttggtaaggt tcttcgcgtt gcatcgaatt 480
aatccgcatg ctccgccgct tgtgcgggcc cccgtcaatt cctttgagtt ttagccttgc 540
ggccgtactc cccaggcggg gcacttaatg cgttagctac ggcgcggaaa acgtggaatg 600
tcccccacac ctagtgccca acgtttacgg catggactac cagggtatct aatcctgttc 660
gctccccatg ctttcgctcc tcagcgtcag taaatgccca gagacctgcc ttcgccatcg 720
gtgttcctcc tgatatctgc gcatttcacc gctacaccag gaattccagt ctcccctaca 780
tcactctagt ctgcccgtac ccaccgcaga tccgaggttg agcctcggac tttcacggca 840
gacgcgacaa accgcctacg agctctttac gcccaataaa tccggataac gcttgcgccc 900
tacgtattac cgcggctgct ggcacgtagt tagccggcgc ttcttctgca ggtaccgtca 960
ctttcgcttc ttccctactg aaagaggttt acaacccgaa ggccgtcatc cctcacgcgg 1020
cgtcgctgca tcaggctttc gcccattgtg caatattccc cactgctgcc tcccgtagga 1080
gtctgggccg tgtctcagtc ccagtgtggc cggtcaccct ctcaggccgg ctacccgtcg 1140
tcgccttggt gagccattac ctcaccaaca agctgatagg ccgcgagtcc atcccccacc 1200
gataaatctt tccaccaaca accatgcggt caaaggtaat atccggtatt agacccagtt 1260
tcccgggctt atcccagagt cgggggcagg ttactcacgt gttactcacc cgttcgccac 1320
taatccaccc a 1331
Claims (5)
1. a kind of Mysore arthrobacterium (Arthrobacter mysorens), which is characterized in that the Mysore arthrobacterium preservation
Title is Mysore arthrobacterium (Arthrobacter mysorens) 7-3, which is preserved in Chinese microorganism strain preservation
Administration committee's common micro-organisms center, the deposit date is on October 17th, 2016, deposit number was CGMCC NO.13118.
2. a kind of application of Mysore arthrobacterium as described in claim 1 in purifying sea water cultivation nitrogenous effluent.
3. a kind of method of purifying sea water cultivation nitrogenous effluent, which is characterized in that by the entitled Mysore arthrobacterium of preservation
The activated seed liquid of (Arthrobacter mysorens) 7-3 is added in the sea-farming nitrogenous effluent that salinity is 30 ‰, in
PH value is 7.0-7.2, shaking table vibrates degradation 48-72h under conditions of 20 DEG C；Mysore arthrobacterium (the Arthrobacter
Mysorens) 7-3 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and the deposit date is 2016 years
October 17, deposit number are CGMCC NO. 13118.
4. the method for purifying sea water cultivation nitrogenous effluent according to claim 3, which is characterized in that the Mysore pole
The activated seed liquid of bacterium (Arthrobacter mysorens) 7-3 the preparation method comprises the following steps: by Mysore arthrobacterium
(Arthrobacter mysorens) 7-3 is inoculated in LB liquid medium, and at 20 DEG C, 150r/min is cultivated for 24 hours, 8000rpm
Thalline were collected by centrifugation, brine three times, resuspended bacterium solution.
5. the method for purifying sea water cultivation nitrogenous effluent according to claim 3 or 4, which is characterized in that the Mysore
The inoculum concentration of the activated seed liquid of arthrobacterium (Arthrobacter mysorens) 7-3 is 5%.
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|---|---|---|---|---|
| CN104862260A (en) * | 2015-06-10 | 2015-08-26 | 国家海洋局第三海洋研究所 | Arthrobacter with aerobic denitrification capability and application thereof |
| CN105713862A (en) * | 2016-03-28 | 2016-06-29 | 武汉科技大学 | Bacterial strain capable of degrading pyridine and ammonia nitrogen, preparation method and application of bacterial strain |
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2016
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104862260A (en) * | 2015-06-10 | 2015-08-26 | 国家海洋局第三海洋研究所 | Arthrobacter with aerobic denitrification capability and application thereof |
| CN105713862A (en) * | 2016-03-28 | 2016-06-29 | 武汉科技大学 | Bacterial strain capable of degrading pyridine and ammonia nitrogen, preparation method and application of bacterial strain |
Non-Patent Citations (2)
| Title |
|---|
| Heterotrophic Nitrification by Arthrobacter sp. (Strain 9006) as Influenced by Different Cultural Conditions, Growth State and Acetate Metabolism;Karl-Paul Witzel 等;《Arch. Microbiol.》;19791231;第137-143页 * |
| Heterotrophic Nitrification by Arthrobacter sp.;W. VERSTRAETE 等;《JOURNAL OF BACTERIOLOGY》;19721231;第955-961页 * |
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