CN109825450A - One plant of resistance to high ammonia nitrogen allotrophic nitrobacteria and its application - Google Patents
One plant of resistance to high ammonia nitrogen allotrophic nitrobacteria and its application Download PDFInfo
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- CN109825450A CN109825450A CN201910046090.0A CN201910046090A CN109825450A CN 109825450 A CN109825450 A CN 109825450A CN 201910046090 A CN201910046090 A CN 201910046090A CN 109825450 A CN109825450 A CN 109825450A
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Abstract
The invention discloses one plant of resistance to high ammonia nitrogen allotrophic nitrobacteria and its applications.The Strain Designation are as follows: allotrophic nitrobacteria (Sphingobacterium sp.) ZFS1-13, deposit number are GDMCC No:60528.The allotrophic nitrobacteria ZFS1-13 well-grown, still holding part ammonia nitrogen removal ability in the simulation high ammonia-nitrogen wastewater that ammonia nitrogen initial concentration is 1500mg/L；In the denitrogenation culture medium that ammonia nitrogen initial concentration is 150mg/L after 180rpm, 28 DEG C of culture 48h, 60% is greater than to the removal rate of ammonia nitrogen, therefore, allotrophic nitrobacteria (Sphingobacterium sp.) ZFS1-13 of the invention can be used for handling ammonia nitrogen waste water, it is resistant to the ammonia nitrogen of high concentration, is of great significance to the development of denitrogenation technology and technique.
Description
Technical field
The present invention relates to microorganism and technical field of environmental science, and in particular to one plant of resistance to high ammonia nitrogen allotrophic nitrobacteria and
It is applied.
Background technique
Currently, water environment pollution problem in China's is got worse.The reason of causing water environment degradation has very much, wherein deriving from
The high ammonia-nitrogen wastewater of landfill leachate, breeding wastewater, coking wastewater etc. is pollution water environment one without direct emission is effectively treated
A major reason.It is flat that high ammonia-nitrogen wastewater input causes lake eutrophication, aquatile death to upset the normal ecosystem
Weighing apparatus, causes to seriously endanger to ecological environment.
Currently, bio-denitrification technology has become one of the Hot Contents of water treatment field research, have inexpensive, efficient
Rate, it is easy to operate, without secondary pollution the advantages that, with development potential.But processing of the current biological denitrogenation technology to high ammonia-nitrogen wastewater
In the presence of very big problem, main cause is that high ammonia-nitrogen wastewater is big to the direct toxic action of microorganism, and microorganism is caused to be difficult in height
Growth is colonized in ammonia nitrogen waste water environment, loses denitrification ability so as to cause it.
Conventionally, nitration reaction is nitrifying process (NH of the autotrophic bacterium under aerobic condition4 +→NH2OH→NO2 -→
NO3 -).With the discovery of allotrophic nitrobacteria, it was recognized that nitration reaction can also occur in heterotroph nitrobacteria.In recent years
Come, many studies have shown that faster, cell yield is higher for the growth rate of allotrophic nitrobacteria compared with Autotrophic nitrification bacterium, needs
The dissolved oxygen concentration wanted is lower, and acid resistance is also stronger.Therefore, novel, efficient allotrophic nitrobacteria is separated from nature, and will
It is applied in the wastewater treatment of high-concentration ammonia-nitrogen, is of great significance to the development of denitrogenation technology and technique.
The allotrophic nitrobacteria having been reported that at present be belonging respectively to Arthrobacter, Flavobacterium,
Achromobacter、Zobellella、Serratia、Pseudomonas、Alcaligenes、Paracoccus、
Bacillus, Enterobacter, Sphingomonas etc. belong to.Sphingobacterium not yet finds allotrophic nitrobacteria at present.
Summary of the invention
The object of the present invention is to provide one plant of allotrophic nitrobacteria (Sphingobacterium sp.) ZFS1-13, and
One kind includes the microbial bacterial agent of the allotrophic nitrobacteria (Sphingobacterium sp.) ZFS1-13, is water body denitrification
A kind of good biomaterial is provided.
Another object of the present invention is to provide the allotrophic nitrobacteria (Sphingobacterium sp.)
The application of ZFS1-13 and corresponding microbial bacterial agent in removal ammonia nitrogen, the especially application in water body removal of ammonia and nitrogen.
To achieve the above object, the invention discloses one plant from the sewage of pig manure water natural aeration pond, and separation screening obtains
Resistance to high ammonia nitrogen allotrophic nitrobacteria, be Sphingobacterium (Sphingobacterium sp.), the entitled ZFS1-13 of bacterial strain,
Belong to Bacteroidetes (Bacteroidetes), sphingolipid Bacteriaceae (Sphingobacteriaceae) on taxology, names are as follows: different
Nitrobacteria (Sphingobacterium sp.) ZFS1-13 is supported, which has been preserved in Guangdong Province's Microbiological Culture Collection
The heart, No. 59 building of compound of address Xianlie Middle Rd., Yuexiu Zone, Guangzhou, Guangdong 100, deposit number are GDMCC No:60528, are protected
The hiding date is on December 20th, 2018.
Allotrophic nitrobacteria (Sphingobacterium sp.) ZFS1-13 of the invention is by inventor in 2 months 2018
It is obtained from pig manure water natural aeration pond sewage sample by 8 months domestication concentration and separations, the bacterial strain physiological and biochemical property are as follows: leather is blue
Family name's negative bacterium, cell shape are rod-shaped, no gemma, atrichia, and a thick layer stickum is wrapped up in thallus periphery, and size is
0.5~1.0 × 0.8~2.0 μm (see Fig. 2)；Bacterium colony is light yellow smooth circle, convexity, neat in edge (see Fig. 1).
The nucleotides sequence of the 16S rRNA of allotrophic nitrobacteria (Sphingobacterium sp.) ZFS1-13 of the invention
Column are as shown in SEQ ID NO:1.In NCBI/GenBank database, 16S rRNA gene order is obtained separated at present
Bacterium in, with Sphingobacterium suaedae strain T47 have highest similitude (94%).
Allotrophic nitrobacteria (Sphingobacterium sp.) ZFS1-13 of the invention is 1500mg/L in ammonia nitrogen concentration
High ammonia nitrogen simulated wastewater in well-grown, when cultivating 48h, OD600 value is up to 0.85, ammonia nitrogen removal frank 12.8%.
Allotrophic nitrobacteria (Sphingobacterium sp.) ZFS1-13 of the present invention is using ammonia nitrogen as the de- of only nitrogen source
In nitrogen culture medium after 180rpm, 28 DEG C of culture 48h, 60% is greater than to the removal rate of nitrate nitrogen, thus, it can be known that the heterotrophism nitre
Changing bacterium ZFS1-13 can be used for handling ammonia nitrogen waste water, removal of ammonia and nitrogen.
Compared with prior art, the invention has the following advantages:
Present invention finds a kind of new allotrophic nitrobacteria (Sphingobacterium sp.) ZFS1-13, the bacterial strains
Well-grown, still holding part ammonia nitrogen removal ability in the simulation high ammonia-nitrogen wastewater that ammonia nitrogen initial concentration is 1500mg/L；In ammonia
Nitrogen initial concentration is to be greater than 60% to the removal rate of ammonia nitrogen in the denitrogenation culture medium of 150mg/L after 180rpm, 28 DEG C of culture 48h,
Therefore allotrophic nitrobacteria (Sphingobacterium sp.) ZFS1-13 of the invention can be used for handling ammonia nitrogen waste water, to de-
The development of nitrogen technology and technique is of great significance.
Allotrophic nitrobacteria (Sphingobacterium sp.) ZFS1-13 of the invention was protected on December 20th, 2018
It is hidden in Guangdong Province's Culture Collection, address is No. 59 building of the compound of Xianlie Middle Rd., Yuexiu Zone, Guangzhou, Guangdong 100,
Deposit number is GDMCC No:60528.
Detailed description of the invention
Fig. 1 is colonial morphology of allotrophic nitrobacteria (Sphingobacterium sp.) ZFS1-13 on solid plate.
Fig. 2 is aobvious in optics after the crystallized purple of allotrophic nitrobacteria (Sphingobacterium sp.) ZFS1-13 dyes
The photo (A) and transmission electron microscope picture (B) of (100 ×) under micro mirror.
Fig. 3 is the representative of allotrophic nitrobacteria (Sphingobacterium sp.) ZFS1-13 and mutually equal interior all categories
The systematic evolution tree of bacterial strain；Wherein, it is contribute using NJ method, only shows that the bootstrap coefficient of > 70% (repeats 1000 in figure
It is secondary).
Fig. 4 is the systematic evolution tree of allotrophic nitrobacteria (Sphingobacterium sp.) ZFS1-13 and close bacterial strain；
Wherein, it is contribute using NJ method, the bootstrap coefficient (repeating 1000 times) of > 70% is only shown in figure.
Fig. 5 is allotrophic nitrobacteria (Sphingobacterium sp.) ZFS1-13 in different initial ammonia nitrogen concentration conditions
Under growth tendency and nitrogen desorption curve schematic diagram.
It in initial ammonia nitrogen concentration is 150mg/L that Fig. 6, which is allotrophic nitrobacteria (Sphingobacterium sp.) ZFS1-13,
Growth tendency and nitrogen desorption curve schematic diagram.
Specific embodiment
With reference to the accompanying drawings and examples, specific embodiments of the present invention will be described in further detail, implements below
Example is not intended to limit the scope of the invention for illustrating the present invention, parameter, ratio of embodiment etc. can adaptation to local conditions make choosing
It selects and substantial effect is had no to result.Unless otherwise noted, method described in embodiment is conventional method, and agents useful for same is equal
The reagent prepared for conventional reagent or in the usual way.
The separation of 1 allotrophic nitrobacteria of embodiment (Sphingobacterium sp.) ZFS1-13
The acquisition of pig manure water natural aeration pond sewage sample is raised pigs from Guangdong Province Shanyi City Jin Ruifeng ecological agriculture Co., Ltd
Field bubble excrement liquid dung level-one natural aeration pond.100mL/ bottles of pig manure water, adds sterilizing pig manure 10g, trained at 30 DEG C, 180rpm
It supports, keeps the skin wet weekly to 100mL；Every two weeks plus 10g sterilize pig manure.Start domestication culture in 2018.02.21,
2018.10.26 separation, acclimating culture about 8 months.Pig manure water sample after taking domestication to cultivate is applied using the dilution of standard
Suspension dilution is applied on Reasoner ' s 2A agar (R2A, Reasoner ' s 2A) plate by cloth method, is placed in 30 DEG C
Culture 3 days；Then bacterium colony and repeatedly scribing line purifying, the bacterium colony of obtained strains ZFS1-13 such as Fig. 1 and Fig. 2 institute are picked from the plate
Show, the bacterium colony be light yellow smooth circle, convexity, neat in edge (Fig. 1), be Gram-negative bacteria, cell shape be it is rod-shaped,
Without gemma, atrichia, a thick layer stickum is wrapped up in thallus periphery, and size is 0.5~1.0 × 0.8~2.0 μm of (figures
2A), transmission electron microscope picture is as shown in Figure 2 B.
Bacterial strain ZFS1-13 after purification makees glycerol tube (- 80 DEG C) and freeze-drying pipe (4 DEG C) preservation respectively.Experiment discovery, bacterial strain
ZFS1-13 generally uses Nutrient Agar or Reasoner ' s 2A agar to cultivate, and cultivation temperature is 28~30 DEG C.
The identification of 2 allotrophic nitrobacteria of embodiment (Sphingobacterium sp.) ZFS1-13
The genomic DNA that bacterial strain ZFS1-13 is extracted using CTAB method, with bacterial 16 S rRNA gene magnification universal primer
27F/1492R (27F:5 '-AGAGTTTGATCCTGGCTCAG-3 ' and 1492R:5 '-TACGACTTAACCCCAATCGC-3 ') expands
Increasing obtains PCR product, and send to Science and Technology Ltd., Shanghai Major Biological Medical Technology Co., Ltd. (Guangzhou Branch) and carry out sequence, sequencing
The 16S rRNA sequence length obtained afterwards is 1401bp, and nucleotide sequence is as shown in SEQ ID NO:1.
The representative strain 16S rRNA gene order for choosing bacterial strain ZFS1-13 and mutually equal interior all categories, uses MEGA
6.0, Kimura 2-parameter model, NJ algorithm build systematic evolution tree (bootstrap is repeated 1000 times), obtained evolution
Tree result is shown in Fig. 3.Then, it chooses bacterial strain ZFS1-13 and close bacterial strain builds systematic evolution tree again in aforementioned manners, finally obtain
Chadogram result see Fig. 4.
In EzBioCloud database, allotrophic nitrobacteria (Sphingobacterium sp.) ZFS1-13 (i.e. bacterial strain
ZFS1-13 16S rRNA gene order) has highest phase with Sphingobacterium suaedae strain T47T
It is next successively Sphingobacterium soli YIM X0211T (93.02%) like property (94.1%),
Sphingobacterium composti 2DSM 18850T (92.92%), Sphingobacterium cellulitidis
R-53603T (92.75%), Sphingobacterium tabacisoli h337T (92.75%).Based on result above point
Analysis, the isolated bacterial strain ZFS1-13 of the Preliminary Identification embodiment of the present invention 1 is Sphingobacterium (Sphingobacterium)
A novel species, suspense name are as follows: allotrophic nitrobacteria (Sphingobacterium sp.) ZFS1-13.
3 allotrophic nitrobacteria of embodiment (Sphingobacterium sp.) ZFS1-13 is resistant to ammonia nitrogen capability evaluation
(1) solution is prepared
Compounded carbons mixed liquor is the preparation method comprises the following steps: take D-Glucose 6.9g, D-Fructose 6.9g, D- lactose 6.9g, sodium acetate
9.5g, mannitol 7.0g, sodium benzoate 4.8g, salicylic acid 4.6g, 90%v/v lactic acid 6.4mL, dehydrated alcohol 7.0mL, glycerol
6.3mL is dissolved in 500mL water, is adjusted pH to 7.4, is used 0.22 μm of membrane filtration degerming.
Multi-vitamins liquid mixed liquor is the preparation method comprises the following steps: take biotin/VH 10mg, folic acid/VB9100mg, pyridoxol/
VB6100mg, thiamine/VB1200mg, niacin 200mg, calcium pantothenate 100mg, VB122mg, riboflavin/VB220mg, lipoic acid
20mg, EDTA-Na 400mg are added in 1L water, are adjusted pH to 7.5, are used 0.22 μm of membrane filtration degerming.
Micro-mixed liquor is the preparation method comprises the following steps: take ZnSO4·7H2O 0.1g、MnCl2·4H2O 0.4g、H3BO3
1.24g、CoCl2·2H2O 0.5g、CuCl2·2H2O 0.5g、NiSO4·6H2O 0.02g、NaMoO4·2H2O 0.4g、KI
0.2g、CaCl2 5.0g、FeSO4·7H2O 1.1g, EDTA-Na 10.0g are dissolved in 1000mL H2O adjusts pH to 7.4, uses
0.22 μm of membrane filtration.
Different ammonia nitrogen concentrations simulate waste water methods are as follows: take sodium succinate 2.5g, trisodium citrate dihydrate 2.5g,
K2HPO41.0g、KH2PO4 1.0g、MgSO4·7H2O 0.2g, is dissolved in 1000mL water, different disposal add respectively 0.471g,
(the NH of 0.942g, 1.884g, 3.297g, 4.710g, 7.065g4)2SO4, i.e., ammonia nitrogen concentration be respectively 100mg/L, 200mg/L,
400mg/L, 700mg/L, 1000mg/L, 1500mg/L adjust pH to 7.4, in 121 DEG C, autoclave sterilization 20min, after cooling
Compounded carbons mixed liquor 1% (v/v) after addition aseptic filtration, multi-vitamins mixed liquor 0.2% (v/v), compound micro member
Plain mixed liquor 0.2% (v/v), mixes to obtain the final product.
(2) it is resistant to ammonia nitrogen capability evaluation
Allotrophic nitrobacteria is accessed in different ammonia nitrogen concentrations simulation sewage by the inoculum concentration of 1%v/v
(Sphingobacterium sp.) ZFS1-13, in 28 DEG C, 180rpm cultivates 48h, then samples, and measures culture solution OD600Value,
Detection ammonia nitrogen concentration (Na Shi reagent spectrophotometry) is simultaneously converted into ammonia nitrogen removal frank, as a result sees Fig. 5.As shown in Figure 5, heterotrophism nitre
It is very strong to high ammonia nitrogen simulation sewage tolerance to change bacterium ZFS1-13, after handling 48h, in the mould that ammonia nitrogen concentration is 1500mg/L
OD in quasi- sewage600Reach 0.85, ammonia nitrogen removal frank 12.8%.
4 allotrophic nitrobacteria of embodiment (Sphingobacterium sp.) ZFS1-13 is trained by only nitrogen source of ammonia nitrogen
The application of nutrient solution
(1) solution is prepared
Preparation of the ammonium ion as only nitrogen source culture medium: simulating the preparation method of sewage referring to examples detailed above 3, will be real
Apply (the NH during the simulation waste water of example 34)2SO4Additional amount be adjusted to 0.707g/L, i.e., ammonia nitrogen concentration is 150mg/L,
Other components and its concentration are consistent.
(2) by the inoculum concentration of 1%v/v in ammonium ion as accessing allotrophic nitrobacteria in only nitrogen source culture medium
(Sphingobacterium sp.) ZFS1-13, is cultivated under 28 DEG C, 180rpm, respectively 0h, 6h, 12h, for 24 hours, 36h,
It is sampled when 48h, 72h, measures culture solution OD600Value, and detect ammonia nitrogen (Na Shi reagent spectrophotometry), nitrite nitrogen (4- amino
Benzsulfamide spectrophotometry) and nitrate nitrogen (ultraviolet spectrophotometry) concentration, as a result see Fig. 6.It will be appreciated from fig. 6 that heterotrophic nitrification
Bacterium ZFS1-13 has removal effect more carefully to ammonia nitrogen in water body, can will be initial dense in water body when the bacterium grows into 48h
Degree is 60% or more the ammonia nitrogen removal of 150mg/L.
The above is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred embodiment is not construed as pair
Limitation of the invention, protection scope of the present invention should be defined by the scope defined by the claims..For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, several improvements and modifications can also be made, these change
It also should be regarded as protection scope of the present invention into retouching.
Sequence table
<110>Guangdong Bo Wote Biotechnology Co., Ltd
<120>one plants of resistance to high ammonia nitrogen allotrophic nitrobacterias and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1400
<212> DNA
<213>allotrophic nitrobacteria ZFS1-13 (Sphingobacterium sp.ZFS1-13)
<400> 1
tgcagtcgac gggatccgcc ggcgagcttg ctcgaaggcg gtgagagtgg cgcacgggtg 60
cgtaacgcgt gagcaacctg cccatatcag ggggatagcc cggcgaaagt cggattaaca 120
ccgcatgaca ataacttacc gcatggtaat ttatttaaat atttatagga tatggatggg 180
ctcgcgtgac attagctagt tggtcggggt aacggcccac caaggctacg atgtctaggg 240
gctctgagag gagaatcccc cacactggta ctgagacacg gaccagactc ctacgggagg 300
cagcagtaag gaatattggt caatgggcgg tagcctgaac cagccatgcc gcgtgcagga 360
tgactgccct atgggttgta aactgctttt gtacgggaac aaccgyctgc tcgtgagcag 420
gcctgagtgt accgtaagaa taaggatcgg ctaactccgt gccagcagcc gcggtaatac 480
ggaggatccg agcgttatcc ggatttattg ggtttaaagg gtgcgtaggc ggccgcttaa 540
gtcaggagtg aaatacggca gctcaactgt cgcagtgctt ttgatactga gtggcttgaa 600
tccatttgaa gtgggcggaa caagacaagt agcggtgaaa tgcatagata tgtcttagaa 660
ccccgattgc gaaggcagct cactaagctg gaattgacgc tgatgcacga aagcgtgggg 720
atcgaacagg attagatacc ctggtagtcc acgccctaaa cgatgatgac tcgatgttag 780
cgatataccg ttagcgtcca agcgaaagcg ttaagtcatc cacctgggga gtacgcccgc 840
aagggtgaaa ctcaaaggaa ttgacggggg cccgcacaag cggaggagca tgtggtttaa 900
ttcgatgata cgcgaggaac cttacccggg cttgaaagtt actgcattat ccagagatgg 960
ataagtcctt cgggacagga aactaggtgc tgcatggctg tcgtcagctc gtgccgtgag 1020
gtgttgggtt aagtcccgca acgagcgcaa cccctatgtt tagttgccag catgtaaagg 1080
tggggactct aaacagactg cctgcgcaag cagtgaggaa ggcggggacg acgtcaagtc 1140
atcatggccc ttacgtccgg ggctacacac gtgctacaat ggacggtaca gcgggcagct 1200
acacagcaat gtggtgcgaa tcccgaaaag ccgttcacag ttcggatcgg ggtctgcaac 1260
tcgaccccgt gaagttggat tcgctagtaa tcgcgtatca gcaatgacgc ggtgaatacg 1320
ttcccgggcc ttgtacacac cgcccgtcaa gccatgaaag ctgggggtgc ctaaagcatg 1380
gccgcaagag cggccagcat 1400
Claims (7)
- Allotrophic nitrobacteria 1. (Sphingobacterium sp.) ZFS1-13, deposit number are as follows: GDMCC No:60528.
- 2. a kind of allotrophic nitrobacteria microbial bacterial agent, which is characterized in that the microbial bacterial agent includes described in claim 1 different Support nitrobacteria (Sphingobacterium sp.) ZFS1-13.
- 3. described in allotrophic nitrobacteria (Sphingobacterium sp.) ZFS1-13 described in claim 1 or claim 2 Allotrophic nitrobacteria microbial bacterial agent removal ammonia nitrogen in application.
- 4. application according to claim 3, which is characterized in that be the application in water body removal of ammonia and nitrogen.
- 5. application according to claim 3, which is characterized in that answered in the ammonia nitrogen waste water ammonia nitrogen removal of enduring high-concentration With.
- 6. application according to claim 5, which is characterized in that the concentration of ammonia nitrogen is in the ammonia nitrogen waste water of the high concentration 1500mg/L。
- 7. application according to claim 3, which is characterized in that allotrophic nitrobacteria (Sphingobacterium sp.) Application of the ZFS1-13 using ammonia nitrogen as only nitrogen source in removal ammonia nitrogen.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111454857A (en) * | 2020-01-08 | 2020-07-28 | 国家林业和草原局竹子研究开发中心 | Efficient nitrifying bacteria and application thereof |
| CN113336336A (en) * | 2021-06-04 | 2021-09-03 | 中国科学院重庆绿色智能技术研究院 | Application of beyerba perniciae in efficient ammonia nitrogen removal |
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| CN109161513A (en) * | 2018-11-13 | 2019-01-08 | 江南大学 | One plant of Sphingobacterium and its application |
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2019
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07274961A (en) * | 1994-04-08 | 1995-10-24 | Toyobo Co Ltd | Production of creatine amidinohydrolase |
| CN106573810A (en) * | 2014-05-14 | 2017-04-19 | 亚拉国际公司 | Denitrification of saline industrial waste water |
| CN109161513A (en) * | 2018-11-13 | 2019-01-08 | 江南大学 | One plant of Sphingobacterium and its application |
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| DHAWAL P.TAMBOLI 等: "Biodegradation of Lignin Monomers Vanillic, p-Coumaric, and Syringic Acid by the Bacterial Strain, Sphingobacterium sp. HY-H", 《JOURNAL OF HAZARDOUS MATERIALS》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111454857A (en) * | 2020-01-08 | 2020-07-28 | 国家林业和草原局竹子研究开发中心 | Efficient nitrifying bacteria and application thereof |
| CN111454857B (en) * | 2020-01-08 | 2021-11-02 | 国家林业和草原局竹子研究开发中心 | Nitrifying bacteria and application thereof |
| CN113336336A (en) * | 2021-06-04 | 2021-09-03 | 中国科学院重庆绿色智能技术研究院 | Application of beyerba perniciae in efficient ammonia nitrogen removal |
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