CN104830724A - Rhizobium strain and application thereof - Google Patents

Rhizobium strain and application thereof Download PDF

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Publication number
CN104830724A
CN104830724A CN201510222556.XA CN201510222556A CN104830724A CN 104830724 A CN104830724 A CN 104830724A CN 201510222556 A CN201510222556 A CN 201510222556A CN 104830724 A CN104830724 A CN 104830724A
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rhizobium
ammonia nitrogen
strain
nitrogen
waste water
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CN104830724B (en
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邹艳艳
梅荣武
韦彦斐
李明智
张宇
王慧荣
许青兰
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Shuiyi Environmental Protection Group Co ltd
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Zhejiang Environmental Science Research and Design Institute
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/41—Rhizobium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • 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
    • C02F3/02—Aerobic processes
    • C—CHEMISTRY; METALLURGY
    • 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
    • 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

Abstract

The invention discloses a rhizobium strain and its application. The rhizobium strain is named as Rhizobium sp., the strain number is zb612, and preservation number is CGMCC No. 10721. The rhizobium strain was preserved in the China General Microbiological Culture Collection Center (CGMCC) at the No.3, 1 West Beichen Road, Chaoyang District of Beijing on April 15th, 2015. The strain provided by the invention can be used in onsite real nitrogen removal from sewage so as to raise the effect of removing ammonia nitrogen from oil sludge. The rhizobium provided by the invention has the characteristic of removing ammonia nitrogen. According to the rhizobium strain, organic carbon can be used as the sole carbon source and ammonia nitrogen is used as the sole nitrogen source so as to carry out metabolism, and ammonia nitrogen is directly converted to N2 through heterotrophic nitrification-aerobic denitrification so as to finish nitrogen removal. Thus, problems of aerobiotic nitrification and anoxic denitrification sectional treatment in traditional biological removal of nitrogen are solved.

Description

One strain rhizobium strains and application thereof
Technical field
The invention belongs to environmental protection technical field, be specifically related to a kind of rhizobium strains and the application thereof with heterotrophic nitrification aerobic denitrifying function.
Background technology
Ammonia nitrogen is one of principal pollutant of water pollution, and ammonia nitrogen exceeds standard and can cause water quality deterioration, body eutrophication, and then causes the major cause of hydrocoles mortality.Ammonia nitrogen degradation can be divided into biological denitrificaion, materialization denitrogenation, but as most economical effective means, biological denitrificaion method also most is promising, but traditional biochemical denitrification approach will through aerobic nitrification and anoxic denitrification two processes, complex process, cost is higher, therefore for the separation of allotrophic nitrobacteria and the application with nitration denitrification function, so to become in biological denitrificaion method an important research contents, there is good development prospect simultaneously.
In recent years, existing scholar isolates efficient ammonia nitrogen degradation bacterium from environment, and is successfully applied in actual sewage, improves original sludge activity and ammonia nitrogen degradation characteristic.Therefore, based on the mechanism of synchronous nitration and denitrification, the separation of nitrification bacteria and being applied in actual sewage is current has important researching value and meaning.
Such as, publication number is that the Chinese invention patent application document of CN 103243050A discloses a strain Low temperature ammonia nitrogen, is applied to Drinking Water at Low Temperature ammonia nitrogen degradation, reaches 107 ~ 109/ml in bacterial strain amount, when initial ammonia nitrogen concentration is 5.0mg/L, with sodium acetate as carbon source, C/N ratio is 2, temperature 2 DEG C, pH value 7.2, under 150rpm (dissolved oxygen is greater than 5mg/L) condition, after ammonia nitrogen degradation 30min, degradation rate can reach 6.36mg NH4+/Lh.
Publication number is the cultural method that the Chinese invention patent application document of CN 104478091A discloses a kind of high-efficiency ammonia nitrogen degradation composite bacteria, (1) get and enter biochemistry pool ammonia nitrogen waste water to be degraded, after filtration, after decolouring, fuel-displaced pre-treatment, 10 times, 8 times, 5 times, 2 times and 0 times of wastewater dilution liquid is prepared into distilled water diluting, for subsequent use; (2) the wastewater dilution liquid culture medium of ammonia nitrogen degradation composite bacteria is prepared; (3) ammonia nitrogen degradation composite bacteria liquid preparation; (4) preserve and activating ammonia nitrogen degradation composite bacteria liquid.Composite bacteria liquid is mainly used in the process of high ammonia-nitrogen wastewater.
Paracoccuspantotrophus is the strain bacterium being found to have heterotrophic nitrification one aerobic denitrification function the earliest, the pathways metabolism that scholar is all over the world special to it expands research extensively and profoundly, about the classification of allotrophic nitrobacteria, degradation kinetics, and the research of zymetology is all being goed deep into.But up to the present, the engineer applied of allotrophic nitrobacteria is also immature.
Bio-denitrification technology is the sewage water denitrification technology of most widespread use, has economy, effectively, and oligosaprobic effect.There is treatment capacity large, cost is low, and do not produce the selection that the advantages such as secondary pollution become everybody, on the basis not changing existing treatment facility, undertaken biological reinforced by adding specific high-effective microorganism bacterium, can improve the removal of original biosystem to target compound, waste water control effect is improved, and thus enjoys everybody to pay attention to and likes.
Summary of the invention
The invention provides strain rhizobium strains and an application thereof, degradation of ammonia nitrogen fast and effectively can be deposited in case in carbon source, providing new approach for synchronously removing ammonia nitrogen.
One strain rhizobium strains, called after root nodule bacterium (Rhizobium sp.), bacterial strain number is zb612, and preserving number is CGMCC No.10721.
Rhizobium strains of the present invention is preserved in the China Committee for Culture Collection of Microorganisms's common micro-organisms center being positioned at No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on April 15th, 2015.
Root nodule bacterium of the present invention (Rhizobium sp.) zb612, belong to gram negative bacterium, sample from the biochemistry pool mud of Refuse Leachate Treatment Plant, acclimating cultivation is carried out in heterotrophic nitrification substratum, ammonia nitrogen, as unique nitrogenous source, obtains finally by dilution plate line separation.
Wherein, the allotrophic nitrobacteria substratum of acclimating bacterial strain: (NH 4) 2sO 40.5g, glucose 5.0g, Vickers salts solution 50ml, is dissolved in water, and supplements distilled water to 1L, regulates pH to 7.0.
Wherein Vickers salts solution: K 2hPO 45.0g, M gSO 47H 2o 2.5g, NaCl 2.5g, FeSO 47H 2o 0.05g, MnSO 44H 2o 0.05g, adds water after dissolving and is settled to 1L.
Bacterial strain provided by the present invention, has following phenotypic characteristic: at 20-40 DEG C, and after LB substratum cultivates 12-24h, bacterium colony is thread, becomes decentralized, and colony colour is white, becomes negative by gramstaining.
The 16S rRNA sequence length of bacterial strain provided by the present invention is 1405bp, and its base sequence is as shown in SEQ ID NO:1.
Root nodule bacterium zb612 provided by the present invention has ammonia nitrogen removal characteristic, and organic carbon can be utilized for sole carbon source, and ammonia nitrogen carries out metabolism as only nitrogen source, is directly N mineralized nitrogen by heterotrophic nitrification-aerobic denitrification effect 2, complete denitrogenation, solve aerobic nitrification in traditional biological denitrogenation, the problem of anoxic denitrification staging treating.
Bacterial strain of the present invention can be used for on-the-spot actual sewage denitrogenation, and to improve the removing ammonia of crude oil mud, therefore, the present invention also provides the application of a kind of rhizobium strains in degrading waste water ammonia nitrogen.
Described waste water is sanitary sewage or pharmacy waste water etc.
During embody rule, the suspension of described root nodule bacterium is added in the Aerobic Pond of waste water treatment process.The dosage of described liquid suspension is 0.5 ~ 1.5% of mud mixture weight in Aerobic Pond.
In described Aerobic Pond, dissolved oxygen controls is 0.2 ~ 1mg/L.Further, dissolved oxygen controls is 0.5 ~ 0.8mg/L.
The pH value of described waste water is 6 ~ 9; Further, the pH value of described waste water is 7 ~ 8.
In described waste water, C/N is 2 ~ 10.
The temperature of described waste water is 25 ~ 40 DEG C; Further, the temperature of described waste water is 25 ~ 35 DEG C.
Root nodule bacterium of the present invention (Rhizobium sp.) zb612, under carbon source exists, can remove ammonia nitrogen and COD, further research finds, it can remove ammonia nitrogen under low dissolved oxygen state, and nitrate-free, the accumulation of nitrite, can be good at the problem overcoming traditional technology.
Bacterial strain of the present invention is applied to the process of high ammonia-nitrogen wastewater, can at low dissolved oxygen, high COD, growth and breeding in high ammonia nitrogen situation, carries out heterotrophic nitrification aerobic denitrifying effect, ammonia nitrogen is converted into nitrogen, nitrate-free, remaining of nitrite, COD also removes simultaneously.When ammonia nitrogen is less than 80mg/L, can almost all degrade completely, ammonia nitrogen be greater than 100 be less than 200 situations under degradation efficiency can reach 50%-76%.
Accompanying drawing explanation
Fig. 1 is the growth curve of root nodule bacterium zb612 in embodiment 1.
Fig. 2 is water outlet ammonia-nitrogen content result figure in embodiment 4.
Embodiment
In embodiment, the method for monitoring and analyzing of various pollutent is with reference to " water and sewage monitoring analytical procedure " (the 4th edition, China Environmental Science Press, 2002), the various units used in embodiment, unified employing national standard.
Ammonia nitrogen degradation medium component: glucose 5g, (NH4) 2sO 40.5g, NaCl 0.3g, K 2hPO 41.0g, MgSO 47H 2o 0.3g, FeSO 47H 2o 0.03g, CaCO 37.5g, adds water and is settled to 1L.
Glucose nitrate culture-medium composition: 5g, glucose, 2g KNO3,1g KH2PO4,1g K2HPO4,0.20g MgSO47H2O, 1 000mL distilled water, pH 7.2 ~ 7.5.
Allotrophic nitrobacteria substratum: (NH 4) 2sO 40.5g, glucose 5.0g, Vickers salts solution 50ml, is dissolved in water, and supplements distilled water to 1L, regulates pH to 7.0.Wherein Vickers salts solution: K 2hPO 45.0g, M gSO 47H 2o 2.5g, NaCl 2.5g, FeSO 47H 2o 0.05g, MnSO 44H 2o 0.05g, adds water after dissolving and is settled to 1L.
Embodiment 1
One, the separation screening of root nodule bacterium zb612
(1) domestication of mud: get certain Refuse Leachate Treatment Plant active sludge 5g, be placed in 250ml ammonia nitrogen degradation substratum, temperature 30 DEG C, rotating speed 100 continuously shaking table cultivates two days, the ammoniumsulphate soln (1.5g/L) of 5ml sterilising treatment is added two days later in Erlenmeyer flask, continue, to its domestication, to repeat this step 3 time, take out muddy water 5ml and leave standstill in the centrifuge tube that sterilizing is good.
(2) Method of Limited Dilution is separated: get the centrifuge tube that above-mentioned supernatant liquor 1ml is placed in 10ml stroke-physiological saline solution, stepwise dilution is to 10 -6,, do three parallel, from 10 -5with 10 -6get the coating of 0.2ml flat board in section, cultivate 48 hours, observe colony growth.
(3) bacterial strain selects purifying: select the good bacterial strain of growing way from flat board and do plate streaking process, wait to grow up to single bacterium colony, is placed in above-mentioned ammonia nitrogen degradation substratum, observes ammonia nitrogen degradation situation, therefrom select ammonia nitrogen degradation rate the highest do further qualification.
Two, the qualification of root nodule bacterium zb612
(1) cultivate form: cultivate 12-24h on LB liquid nutrient medium after, bacterium colony is thread, become decentralized, colony colour is white, and solid medium substratum middle plateform was cultivated after 24-36 hour, and bacterium colony is rounded, and protruding, there is rete malpighii on surface.
(2) gramstaining: be Gram-negative bacteria by gramstaining.
(3) 16s rRNA identifies: solid slope is delivered to Sangon Biotech (Shanghai) Co., Ltd. and be 16s rRNA and identify, sequence is as shown in SEQ ID NO:1, and qualification result is root nodule bacterium.
By above qualification result, confirm that above-mentioned bacterial strains is the Gram-negative bacteria of rhizobium, by its called after root nodule bacterium (Rhizobium sp.), bacterial strain number is zb612, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preserving number is: CGMCC No.10721, the preservation time: on April 15th, 2015.
Embodiment 2
The growth of root nodule bacterium Rhizobium sp.zb612 and ammonia nitrogen degradation measure
Be inoculated in by root nodule bacterium Rhizobium sp.zb612 in the LB liquid nutrient medium (yeast extract paste 5g/L, peptone 10g/L, NaCl 10g/L) of 100ml, in 30 DEG C, 100rpm, shaking table vibration is cultivated.After cultivating 48h, thalline packing be stored in the good preservation pipe of sterilizing, each preservation pipe adds 0.5ml bacterium liquid, 0.5ml30% glycerine.Negative 80 DEG C of preservations, for future use.
Bacterial strain being chosen a ring is inoculated in 100ml allotrophic nitrobacteria substratum, 30 DEG C, 100rpm, shaking culture 24h, as the Active vaccination liquid of formal degradation experiment, get Active vaccination liquid 5% and carry out 2 groups of experiments respectively, often organizing access three respectively contains in the good ammonia nitrogen degradation substratum of 100ml sterilizing (concentration of ammonia nitrogen is 150ml/L), in 30 DEG C, 100rpm, shaking culture, wherein one group, every several hours sampling and measuring thalline optical density(OD), draw out thalli growth curve, after another group 48H days, measure ammonia nitrogen residual concentration.
Thalli growth curve is as shown in Figure 1: root nodule bacterium zb612 growth adaptation phase section, the growth of logarithmic phase can be entered into very soon, logarithmic phase is 50h hour to time stationary phase, enters into the paracme afterwards, and reason may be that culture medium carbon source approach exhaustion causes thalline to continue merisis.After thalli growth 48 hours, ammonia nitrogen degradation reaches 86.6%.
Embodiment 3
The aerobic denitrification test of root nodule bacterium Rhizobium sp.zb612
In the Erlenmeyer flask of 500ml, add 200ml glucose nitrate culture-medium, thallus suspension liquid adds by 2% volume, and shaking table cultivates 48 hours, observes nitrate residual quantity.
Test-results shows: after 48h, and the residual quantity of nitrate concentration is 48.4mg/L, the accumulation that nitrite is little, and former water nitrate concentration 352.7mg/L, degradation rate is: 86.2%.
Test-results shows, under aerobic condition, root nodule bacterium zb612 has the function of aerobic denitrification, does not almost want the accumulation of nitrite simultaneously.
Experimental example 4
The actual sewage denitrogenation lab scale research of root nodule bacterium zb612
In the packaged unit of simulated field waste disposal plant, the liquid suspension of thalline adds by the amount of 1% volume, adds the active sludge of 2% volume simultaneously, water inlet NH 3-N1 is 160mg/l, COD is 2000, and dissolved oxygen controls between 0.5-0.8mg/L, and return time is 2 days, and within every two days, measure water outlet ammonia-nitrogen content, experiment runs 3 months continuously
Experimental result is as shown in Figure 2: water outlet ammonia nitrogen is stablized as figure shows, maintains 10-30mg/l.It can thus be appreciated that: root nodule bacterium provided by the invention, under low dissolved oxygen state, can be good at carrying out nitration denitrification, and effect stability.

Claims (9)

1. a strain rhizobium strains, is characterized in that, called after root nodule bacterium (Rhizobium sp.), and bacterial strain number is zb612, and preserving number is CGMCC No.10721.
2. the application of rhizobium strains in degrading waste water ammonia nitrogen as claimed in claim 1.
3. apply according to claim 2, it is characterized in that, described waste water is sanitary sewage or pharmacy waste water.
4. apply according to claim 2, it is characterized in that, comprising: the suspension of described root nodule bacterium is added in the Aerobic Pond of waste water treatment process.
5. apply according to claim 4, it is characterized in that, the dosage of described liquid suspension is 0.5 ~ 1.5% of mud mixture weight in Aerobic Pond.
6. apply according to claim 4, it is characterized in that, in described Aerobic Pond, dissolved oxygen controls is 0.2 ~ 1mg/L.
7. apply according to claim 4, it is characterized in that, the pH value of described waste water is 6 ~ 9.
8. apply according to claim 4, it is characterized in that, in described waste water, C/N is 2 ~ 10.
9. apply according to claim 4, it is characterized in that, the temperature of described waste water is 25 ~ 40 DEG C.
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105110561A (en) * 2015-08-28 2015-12-02 浙江省环境保护科学设计研究院 Method for treating high ammonia-nitrogen wastewater under low dissolved oxygen condition
CN105713864A (en) * 2016-04-15 2016-06-29 中国科学院东北地理与农业生态研究所 Rapid scribing and separating method for nodule bacteria
CN105906151A (en) * 2016-06-08 2016-08-31 浙江省环境保护科学设计研究院 Sewage treatment device and method of high-density biochemical system in combination with activated carbon filtration system
CN105925516A (en) * 2016-07-13 2016-09-07 广东省微生物研究所 Facultatively autotrophic sulfur oxidation denitrification rhizobium F43b and application thereof
CN106167774A (en) * 2016-07-06 2016-11-30 四川省原子能研究院 Root nodule bacteria Scot1305 and the application in promoting plant soil restoration heavy metal pollution thereof
CN107043169A (en) * 2016-02-05 2017-08-15 三菱化学株式会社 Wastewater treatment equipment and method of wastewater treatment

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105110561A (en) * 2015-08-28 2015-12-02 浙江省环境保护科学设计研究院 Method for treating high ammonia-nitrogen wastewater under low dissolved oxygen condition
CN105110561B (en) * 2015-08-28 2017-12-12 浙江省环境保护科学设计研究院 A kind of method that high ammonia-nitrogen wastewater is handled under low dissolved oxygen condition
CN107043169A (en) * 2016-02-05 2017-08-15 三菱化学株式会社 Wastewater treatment equipment and method of wastewater treatment
CN105713864A (en) * 2016-04-15 2016-06-29 中国科学院东北地理与农业生态研究所 Rapid scribing and separating method for nodule bacteria
CN105906151A (en) * 2016-06-08 2016-08-31 浙江省环境保护科学设计研究院 Sewage treatment device and method of high-density biochemical system in combination with activated carbon filtration system
CN106167774A (en) * 2016-07-06 2016-11-30 四川省原子能研究院 Root nodule bacteria Scot1305 and the application in promoting plant soil restoration heavy metal pollution thereof
CN106167774B (en) * 2016-07-06 2019-05-10 四川省原子能研究院 Rhizobium Scot1305 and its application in promotion plant soil restoration heavy metal pollution
CN105925516A (en) * 2016-07-13 2016-09-07 广东省微生物研究所 Facultatively autotrophic sulfur oxidation denitrification rhizobium F43b and application thereof
CN105925516B (en) * 2016-07-13 2019-08-09 广东省微生物研究所 A kind of facultative autotrophy type sulphur oxidation denitrification rhizobium F43b and its application

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