CN105154349B - A kind of heterotrophic nitrification Facultative Halophiles and its application - Google Patents
A kind of heterotrophic nitrification Facultative Halophiles and its application Download PDFInfo
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- CN105154349B CN105154349B CN201510390281.0A CN201510390281A CN105154349B CN 105154349 B CN105154349 B CN 105154349B CN 201510390281 A CN201510390281 A CN 201510390281A CN 105154349 B CN105154349 B CN 105154349B
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- heterotrophic nitrification
- facultative halophiles
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
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Abstract
The invention belongs to technical field of biological environmental protection, and in particular to a kind of heterotrophic nitrification Facultative Halophiles and its application.The Strain Designation is Hydrogenophaga pseudoflava sp.C2, belong to class yellow food hydrogen production water bacterium, China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC) is preserved on June 10th, 2015, deposit number is:CGMCC No.10977.Heterotrophic nitrification Facultative Halophiles of the present invention may be directly applied in domestic sewage in rural areas improvement, have preferable denitrification effect.By adding the bacterium, nitrogen and CODcr removal efficiency in the domestic sewage in rural areas high to salt content can be effectively improved.
Description
Technical field
The invention belongs to technical field of biological environmental protection, and in particular to a kind of heterotrophic nitrification Facultative Halophiles and its application.
Background technology
There is administrative village more than 60 ten thousand in China at present, and village population has 7.6 hundred million, wherein 96% village lacks sewage disposal system
System.Country sewage radix is big, but is influenceed by the development level such as Rural Economic Development Levels, production, life style, different regions agriculture
The sewage discharge characteristic difference in village is very big.The technology and operational mode that urban wastewater treatment firm uses at present are not suitable for rural area
The processing of decentralized sewage.As country puts more effort to rural development, Peasants Joy, village industry and breeding enterprise etc. are no
Disconnected to increase, country sewage type is continuously increased, and salt content, nitrogen phosphorus equal size constantly raise in sewage, in addition chemical fertilizer and agricultural chemicals
Use, which increase waste water control difficulty.The unordered discharge of sewage causes the pollution of surface water, underground water, reservoir, pond and river course
It is serious Deng water body blackening, eutrophication, odor pollution air, have a strong impact on living environment and physical and mental health.In addition, rural area
Economy more falls behind, and highly energy-consuming, the technology compared with high cost are difficult popularization and application.Currently, it is necessary to find a kind of cost it is relatively low, without two
Secondary pollution, have a wide range of application and adaptable technology, and be adapted in Rural Areas of China popularization and application.
With a large amount of introductions of the high speed development of China's rural economy, the particularly tertiary industry, the type of sewage is not yet
Disconnected increase, wherein brine waste are distributed the most extensive, special food and drink waste water, seriously govern the denitrogenation scavenging effect of denitrifier.
Therefore, people still suffer from demand for that can carry out the mushroom of rural sewage treatment.
The content of the invention
It is an object of the invention to provide a kind of heterotrophic nitrification Facultative Halophiles and its application.The heterotrophic nitrification Facultative Halophiles are from lake
Separation screening is obtained in northern Shiyan domestic sewage in rural areas, and the bacterium can be applied in sewage disposal.
Heterotrophic nitrification Facultative Halophiles provided by the invention, the bacterium are named as Hydrogenophaga pseudoflava
Sp.C2, belong to class yellow food hydrogen production water bacterium, China Committee for Culture Collection of Microorganisms is preserved on June 10th, 2015
Common micro-organisms center (abbreviation CGMCC;Address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences microorganism
Research institute), deposit number is:CGMCC No.10977.
Preferably, for the 16S rDNA sequences of the heterotrophic nitrification salt tolerant bacteria strain as shown in sequence table 1, its length is 1372bp.
Preferably, the heterotrophic nitrification salt tolerant bacteria strain has following phenotypic characteristic:At 30~37 DEG C, agar medium
After 24~72h of upper culture, bacterium colony surface polishes, faint yellow;Thalline thin rod shape, it is 0.3 × 0.6 μm~1.5 μm, single, paired
Arrange, raw in gemma, ellipse, sporangiocyst expands, Gram-negative.
According to the morphological feature of the heterotrophic nitrification Facultative Halophiles and physiological and biochemical property and its 16S rDNA in Genbank
Retrieval result, and drawing system development tree, the comprehensive identification bacterial strain is that class yellow eats hydrogen and produces water bacterium and (is named as
Hydrogenophaga pseudoflava sp.C2)。
Preferably, the tolerance of salinity (in terms of NaCl) of the heterotrophic nitrification salt tolerant bacteria strain is 1%~10 weight %, more preferably
The weight % of ground 1%~3.
Preferably, the removal ammonia nitrogen effect of the heterotrophic nitrification salt tolerant bacteria strain is less than 200mg/L for starting ammonia nitrogen concentration,
More preferably 0~100mg/L.
The present invention provides application of the above-mentioned heterotrophic nitrification Facultative Halophiles in sewage purification.
The salt content (in terms of NaCl) of the sewage is 1%~10 weight %, and starting ammonia nitrogen concentration is less than 200mg/L.
Preferably, the salt content (in terms of NaCl) of the sewage is 1%~3 weight %, and starting ammonia nitrogen concentration is 0~100mg/L.
Especially, the sewage is domestic sewage in rural areas.
Heterotrophic nitrification Facultative Halophiles (Hydrogenophaga pseudoflava sp.C2, the food hydrogen production of class yellow of the present invention
Water bacterium) have the advantages that compared with the prior art:
(1) heterotrophic nitrification salt tolerant bacteria strain of the invention is under aerobic condition, under conditions of salt content height, using ammonia nitrogen as only
One nitrogen source, carry out heterotrophic nitrification.Using this characteristic of the bacterial strain, the high rural area life of salt content can be directly applied to
In sewage living, such as in food waste sewage etc., it may be implemented in the removal to nitrogen in the high sewage of salt content.Thus, its application prospect
It is preferable with social benefit.
(2) tolerance of salinity (in terms of NaCl) of heterotrophic nitrification salt tolerant bacteria strain of the invention is 1%~10 weight %, in salinity
1%~3 weight % scope can grow and efficient denitrification.
(3) heterotrophic nitrification salt tolerant bacteria strain of the invention can play in scope of the starting ammonia nitrogen concentration less than 200mg/L
Denitrification functions are higher in 0~100mg/L denitrification percents.
The heterotrophic nitrification Facultative Halophiles of the present invention are the indigenous microorganism in the domestic sewage in rural areas high screened from salt content, will
It is used in the high domestic sewage in rural areas of salt content, its economic benefits, it will greatlys save cost of sewage disposal, is also not required to
Consider the security of strain, be adapted to the rural area more scattered on China aggregation ground to use.Heterotrophic nitrification salt tolerant of the present invention
Bacteria strain has preferable denitrification effect, by adding the bacterium, can effectively improve the indigenous microorganism life in the countryside high to salt content
Nitrogen and CODcr removal efficiency in sewage.
Brief description of the drawings
Fig. 1 is heterotrophic nitrification Facultative Halophiles of the present invention (Hydrogenophaga pseudoflava sp.C2) bacterial strain thalline shape
State electromicroscopic photograph.
Fig. 2 is heterotrophic nitrification Facultative Halophiles of the present invention (Hydrogenophaga pseudoflava sp.C2) bacterial strain bacterium colony shape
State photo.
Fig. 3 be heterotrophic nitrification Facultative Halophiles of the present invention (Hydrogenophaga pseudoflava sp.C2) bacterial strain to it is related
The 16S rDNA sequences development tree of kind.
Fig. 4 is the growth curve of heterotrophic nitrification Facultative Halophiles of the present invention (Hydrogenophaga pseudoflava sp.C2) bacterial strain.
Fig. 5 is heterotrophic nitrification Facultative Halophiles of the present invention (Hydrogenophaga pseudoflava sp.C2) bacterial strain in difference
Originate the denitrification effect under ammonia nitrogen concentration.
Fig. 6 is heterotrophic nitrification Facultative Halophiles of the present invention (Hydrogenophaga pseudoflava sp.C2) bacterial strain in difference
Denitrification effect under salinity.
Embodiment
The separation identification of 1 bacterial strain of the present invention of embodiment:
(1) culture medium:
1. denitrogenation culture medium:Ammonium sulfate 0.48g, butanedioic acid 4.71g, trace element solution 50mL, distilled water is used after dissolving
1L is settled to, pH is controlled 7.0~7.2.(solid medium, adding 2% agar)
The trace element solution:K2HPO4·3H2O 6.5g, MgSO4·7H2O 2.5g, NaCl 2.5g, FeSO4·
7H2O 0.05g, MnSO4·H2O 0.04g, 1L is settled to after dissolving with distilled water;
2. resistance to salt culture medium:5g/L yeast extract powder, 10g/L tryptones, 50g/L NaCl, pH 7.0~7.5;
(2) separation screening of denitrogenation bacterial strain
10mL Hubei Shiyan domestic sewage in rural areas is taken with Sterile pipette, the triangular flask that capacity is 250mL is placed in and (wherein contains
Have the sterile denitrogenation culture mediums of 100mL) in, shaking table enrichment culture, condition of culture is 30 DEG C, rotating speed 160rpm, each to be commissioned to train foster three
My god.Using the presence of diphenylamines reagent detection Nitromonas.In culture medium until there is Nitromonas in enrichment culture bacterium solution.
Nutrient solution containing Nitromonas is carried out by flat board coating using the method for Method of Limited Dilution, trained in 37 DEG C of constant incubators
After supporting 48h, choose single bacterium colony and carry out multiple flat board sectional streak.When observing no miscellaneous bacteria under the microscope, bacterial strain purifying terminates.
Purified bacterial strain is subjected to ammonia nitrogen removal screening experiment, bacterial strain is seeded in resistance to salt culture medium respectively, 37 DEG C of constant temperature
Cultivate after cultivating 48h in phase, determine ammonia nitrogen removal frank, choose bacterial strain of the ammonia nitrogen removal frank higher than 85% as the denitrifier chosen.
(3) screening of denitrogenation salt-enduring strain
The denitrogenation bacterial strain screened is subjected to Method of Limited Dilution respectively and flat board is coated on resistance to salt culture medium.It can be trained in salt tolerant
It is denitrogenation Facultative Halophiles to support well-grown bacterial strain on base.
(4) one plant of denitrogenation Facultative Halophiles is screened, is characterized as:At 30~37 DEG C, 24~72h is cultivated on agar medium
Afterwards, bacterium colony surface polishes, faint yellow;It is negative under the microscope after Gram's staining, thalline is in thin rod shape, and 0.3 × 0.6
μm~1.5 μm, single, paired arrangement is raw in gemma, and ellipse, sporangiocyst expands.It is specifically shown in Fig. 1 and Fig. 2.Fig. 1 is different for the present invention
Support nitrification salt tolerant bacteria strain thalli morphology electromicroscopic photograph.Fig. 2 is heterotrophic nitrification salt tolerant bacteria strain colonial morphology photo of the present invention.
(5) 16S rDNA sequence analyses and Phylogenetic Analysis
By 16S rDNA PCR amplifications and sequencing, obtain 16S rDNA gene orders length and (seen below for 1372bp
Sequence table 1).Submit Genbank to be compared, find heterotrophic nitrification Facultative Halophiles C2 and Hydrogenophaga of the present invention
Pseudoflava homologys can reach 99%.Using the softwares of MEGA 5.0, ortho position connection method shows the bacterial strain and related kind
16S rDNA sequences development tree, carries out the similarity of 1000 times and computes repeatedly, and tree node is developed in figure and only shows Bootstrap values
More than 50% numerical value (see Fig. 3.Fig. 3 is that heterotrophic nitrification salt tolerant bacteria strain of the present invention is developed to the 16S rDNA sequences of related kind
Tree).It is Hydrogenophaga pseudoflava sp.C2 to name the bacterial strain, and is preserved in China on June 10th, 2015
Microbiological Culture Collection administration committee common micro-organisms center (abbreviation CGMCC), preserving number are CGMCC No.10977.
The growth curve determination experiment of embodiment 2
Take 18 sterile Boiling tubes, incubation time indicated respectively with marking pen, i.e., 0,2,4,6,8,10,12,14,16,18,
20th, 22,24,26,28,30,32 and 34h.
The heterotrophic nitrification Facultative Halophiles Hydrogenophaga obtained in 2.5mL embodiments 1 is drawn with sterile tube
Pseudoflava sp.C2 nutrient solutions are transferred in the triangular flask for filling 250mL denitrogenation culture mediums, and 8mL is taken respectively after well mixed
Mixed liquor is transferred in 18 sterile Boiling tubes of above-mentioned mark.Vaccinated test tube is put into 37 DEG C of concussion and cultivates of shaking table, rotating speed
160rpm, 0,2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32 and 34h is cultivated respectively.It will indicate corresponding
The test tube of time takes out, and is immediately placed in refrigerator, to be measured.Blank control is done with nonvaccinated denitrogenation nutrient solution, will before sample determines
Nutrient solution fully vibrates, and is uniformly distributed cell.Photoelectricity turbidimetric assay is carried out from 600nm wavelength.Specific measurement result is shown in figure
4.Fig. 4 is the growth curve of heterotrophic nitrification Facultative Halophiles of the present invention (Hydrogenophaga pseudoflava sp.C2) bacterial strain.
As seen from Figure 4, heterotrophic nitrification Facultative Halophiles (Hydrogenophaga pseudoflava sp.C2) of the present invention
Well-grown, 0~4h are in the laundering period of bacterial strain, and 5~26h enters logarithmic growth, and 27~32h is the stationary phase of bacterial strain, after 33h
Into decline phase.
The tolerance of salinity determination test of embodiment 3
Using the method for dilution spread, the heterotrophic nitrification Facultative Halophiles (Hydrogenophaga of the present invention that will have been activated
Pseudoflava sp.C2) bacterium solution is coated on salt tolerant solid medium, salinity (being calculated with NaCl) is respectively 1%, 3%,
5%th, 8%, the dilution factor for activating bacterium solution is 10-5、10-4、10-3、10-2、10-1, the culture medium that does not connect strain is blank control, 30
DEG C, constant incubator culture 2 days, carry out plate count.As a result it see the table below 1.
From the result in table 1 below, heterotrophic nitrification Facultative Halophiles (Hydrogenophaga pseudoflava of the present invention
Sp.C2) can be grown in the high environment of salt content, the tolerance of salinity (in terms of NaCl) be 1%~8%, the most suitable tolerance of salinity be 1~
3%, cfu can reach 7~9 × 10-6。
Survival of the bacterial strain of table 1 under the different tolerance of salinitys
Salinity | 1% | 3% | 5% | 8% |
cfu | 9×106 | 7×106 | 4×104 | 1×102 |
The denitrification effect of embodiment 4 is tested
By heterotrophic nitrification Facultative Halophiles of the present invention (Hydrogenophaga pseudoflava sp.C2) inoculation in
In the denitrogenation culture medium of 100mL different nitrogen contents, starting ammonia nitrogen concentration is respectively 50mg/L (processing one), 100mg/L (processing
Two), 200mg/L (processing three), the culture medium for not connecing strain are blank control, and 37 DEG C, rotating speed 160rpm, shaking table culture case is trained
Support 30h.Take nutrient solution to be centrifuged 6 minutes under 10000rpm respectively, by 0.45 μm of membrane filtration, determine the ammonia in nutrient solution
The content of nitrogen and nitrate nitrogen.Specific measurement result is shown in Fig. 5.Fig. 5 is heterotrophic nitrification Facultative Halophiles (Hydrogenophaga of the present invention
Pseudoflava sp.C2) denitrification effect of the bacterial strain under different starting ammonia nitrogen concentrations.
As seen from Figure 5, heterotrophic nitrification Facultative Halophiles (Hydrogenophaga pseudoflava sp.C2) of the present invention
Bacterial strain is larger in different starting ammonia nitrogen removal frank differences, and the clearance of three processing is respectively 98%, 90% and 79%, and nitre
Sour nitrogen is accumulative less, illustrates that heterotrophic nitrification salt tolerant bacteria strain of the present invention has preferable heterotrophic nitrification function, it is dense to originate ammonia nitrogen
Scope of the degree less than 200mg/L can play denitrification functions, and when originating ammonia nitrogen concentration and being less than 100mg/L, best results.
The different salinity denitrogenation degradation experiment of embodiment 5
By heterotrophic nitrification Facultative Halophiles of the present invention (Hydrogenophaga pseudoflava sp.C2) inoculation in
In the denitrogenation culture medium of 100mL differences saliferous (in terms of NaCl), salinity is respectively 1 weight % (processing one), 3 weight % (processing
Two), 5 weight % (processing three) and 8 weight % (processing three), starting ammonia nitrogen concentration is 50mg/L, and the culture medium for not connecing strain is
Blank control.37 DEG C, rotating speed 160rpm, shaking table culture case culture 30h.Nutrient solution is taken to be centrifuged 6 minutes under 10000rpm respectively,
By 0.45 μm of membrane filtration, the content of the ammonia nitrogen and nitrate nitrogen in nutrient solution is determined.Specific measurement result is shown in Fig. 6.Fig. 6 is this hair
Bright heterotrophic nitrification Facultative Halophiles (Hydrogenophaga pseudoflava sp.C2) denitrification effect of the bacterial strain under different salinity.
As seen from Figure 6, heterotrophic nitrification Facultative Halophiles (Hydrogenophaga pseudoflava sp.C2) of the present invention
For bacterial strain under different salinity, ammonia nitrogen removal frank difference is larger, and the clearance of four processing is respectively 88%, 84%, 76% and
66%, and nitrate nitrogen is accumulative less, illustrates that heterotrophic nitrification salt tolerant bacteria strain of the present invention has preferable heterotrophic nitrification salt resistant function,
When originating ammonia nitrogen concentration 50mg/L, the preferred tolerance of salinity of heterotrophic nitrification Facultative Halophiles is 1%~3%.
The denitrogenation salt tolerant domestic sewage in rural areas degradation experiment of embodiment 6
(1) waste water processing early stage:Tianjin Yuqiao Reservoir Peasants Joy waste water is taken, and by the grid of 30 mesh, goes to remove water
Larger-diameter suspension in body.By detection:Wastewater pH 7.2, ammonia nitrogen concentration 70.6mg/L, CODcr 320mg/L.Together
When NaCl is added into waste water, salt content in waste water is maintained 3%.
(2) C2 bacterium solutions are directly added in the treated waste water of 5L, inoculum concentration is 10% volume.Cultivation temperature is 30
DEG C, sewage purification process is without pH in control targe water body, mixing speed 160rpm.As a result:After processing 4 days, ammonia in water body
The clearance of nitrogen can reach 78.4%, CODcr clearances and can reach 57.2%.
Claims (9)
1. a kind of heterotrophic nitrification Facultative Halophiles, the bacterium is named as Hydrogenophaga pseudoflava sp.C2, belongs to class Huang
Color food hydrogen production water bacterium, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on June 10th, 2015,
Deposit number is:CGMCC No.10977.
2. heterotrophic nitrification Facultative Halophiles according to claim 1, it is characterised in that the heterotrophic nitrification salt tolerant bacteria strain
16S rDNA sequences are as shown in sequence table 1.
3. heterotrophic nitrification Facultative Halophiles according to claim 1, it is characterised in that the heterotrophic nitrification salt tolerant bacteria strain has
Following phenotypic characteristic:At 30~37 DEG C, after cultivating 24~72h on agar medium, bacterium colony surface polishes, faint yellow;Bacterium
Body thin rod shape, 0.3 × 0.6 μm~1.5 μm, single, paired arrangement is raw in gemma, and ellipse, sporangiocyst expands, Gram-negative.
4. heterotrophic nitrification Facultative Halophiles according to claim 1, it is characterised in that the heterotrophic nitrification salt tolerant bacteria strain with
The tolerance of salinity of NaCl meters is 1%~8 weight %.
5. heterotrophic nitrification Facultative Halophiles according to claim 4, it is characterised in that the heterotrophic nitrification salt tolerant bacteria strain with
The tolerance of salinity of NaCl meters is 1%~3 weight %.
6. the heterotrophic nitrification Facultative Halophiles according to claim 4 or 5, it is characterised in that the heterotrophic nitrification salt tolerant bacteria strain
Removal ammonia nitrogen effect for starting ammonia nitrogen concentration be less than 200mg/L.
7. heterotrophic nitrification Facultative Halophiles according to claim 6, it is characterised in that the heterotrophic nitrification salt tolerant bacteria strain is gone
Except ammonia nitrogen effect is that starting ammonia nitrogen concentration is 0~100mg/L.
8. application of the heterotrophic nitrification Facultative Halophiles in domestic sewage in rural areas purification described in claim 1, the life in the countryside are dirty
Water using the salt content that NaCl is counted as 1%~8 weight %, starting ammonia nitrogen concentration be less than 200mg/L.
9. application according to claim 8, it is characterised in that the salt content in terms of NaCl of the domestic sewage in rural areas is
1%~3 weight %, starting ammonia nitrogen concentration are 0~100mg/L.
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CN109112080B (en) * | 2018-07-07 | 2020-05-12 | 华中农业大学 | Cytophaga hygrophila H7 with aromatic compound degradation, nitrogen removal and arsenic removal capabilities and application |
CN110540302A (en) * | 2019-05-31 | 2019-12-06 | 广东五色时空生态环境科技有限公司 | Pollution source gridding biological intervention pollution-reducing deodorizing process |
CN110628670B (en) * | 2019-09-18 | 2020-12-04 | 自然资源部第三海洋研究所 | Heterotrophic nitrifying bacteria and application thereof |
CN113249273B (en) * | 2021-06-30 | 2022-03-08 | 泸州老窖股份有限公司 | Salt-tolerant bacillus subtilis and application thereof in high-salt ammonia nitrogen wastewater treatment |
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