CN102465103B - Aerobic denitrification methylobacterium phyllosphaerae and application thereof - Google Patents
Aerobic denitrification methylobacterium phyllosphaerae and application thereof Download PDFInfo
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- CN102465103B CN102465103B CN2010105361690A CN201010536169A CN102465103B CN 102465103 B CN102465103 B CN 102465103B CN 2010105361690 A CN2010105361690 A CN 2010105361690A CN 201010536169 A CN201010536169 A CN 201010536169A CN 102465103 B CN102465103 B CN 102465103B
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Abstract
The invention relates to an aerobic denitrification methylobacterium phyllosphaerae strain and application thereof. The aerobic denitrification strain is methylobacterium phyllosphaerae SDN-3CGMCC No.3660. The strain can be used for carrying out aerobic denitrification by using nitrate nitrogen and can also be used for carrying out hetetrophic nitrification-aerobic denitrification by using ammonia nitrogen, under aerobic and limited-oxygen conditions. The strain is stable in genetic character, and can be used for realizing synchronous nitrification-denitrification and independently completing the whole process of biological denitrification. The strain is used for treating various nitrogen-containing wastewater by needing only one aerobic stage, the process is simple and the denitrification efficiency is high.
Description
Technical field
The invention belongs to the environmental microorganism field, be specifically related to methyl bacillus (Methylobacterium phyllosphaerae) and application thereof that a strain is used for biological denitrificaion.Methyl bacillus of the present invention all has stronger denitrification capability under aerobic and limited oxygen condition, be specially adapted to the denitrogenation of ammonia-containing water synchronous nitration and denitrification process.
Background technology
Along with the pollution of Water element is day by day serious, denitrogenation becomes emphasis and the focus of present water treatment research.Generally believe at present, biological denitrificaion is one of comparatively cost-effective method of removing from waste water nitrate pollution.The traditional biological denitrogenation comprises aerobic nitrification and two processes of anaerobic denitrifying, and this just need to separate nitrification and denitrification, exists technique tediously long, and the sewage treatment structure floor space is large, many drawbacks such as investment and working cost height.For the problems referred to above, some investigator attempts nitrifier and denitrifying bacteria are added in the same reactor, although part has solved the problem that traditional technology exists, because the difference of two kinds of thalline, the total nitrogen removal effect is often undesirable.
So people constantly seek novel, efficient denitrification microorganism, aerobic denitrifying bacteria receives much attention as a kind of novel denitrifying bacteria.In recent years, the aerobic denitrifying bacteria of having found has general foster sulphur coccus (Thiosphaera pantotropha), now rename Paracoccus denitrificans (Paracoccus denitrificans) as, Bacillus foecalis alkaligenes (Alcaligenes faecalis), marine pseudomonas (Pseudomonas nautical) and tiny aerobic denitrifying bacteria (Microvirgula aerodenitrificans) etc.The discovery of this type of bacterial strain has broken through the conventional art restriction, has greatly simplified the denitrification process link, can realize the Simultaneous Nitrification denitrification denitrogenation.
CN200910083014.3 discloses the edaphic bacillus (Agrobacterium sp.) that a strain has heterotrophic nitrification-aerobic denitrification capability.This bacterium is being processed take organism (sodium succinate, Trisodium Citrate and anhydrous sodium acetate etc.) as carbon source, and when ammonia nitrogen was the waste water of only nitrogen source, ammonia nitrogen removal frank 80%～100% was removed speed 3.14mg/ (Lh)～7.95mg/ (Lh); Nitrogen removal rate 78%～86.54%.When the waste water of processing take nitrate nitrogen or nitrite nitrogen as only nitrogen source, nitrate nitrogen clearance 64.05%～100% is removed speed 3.12mg/ (Lh)～5.44mg/ (Lh); Nitrogen removal rate 63.28%～71.25%.Zhang Guangya etc. (using and the environmental organism journal 2005,11 (2)) are separated to a strain aerobic simultaneous nitrification ﹠ denitrification bacterium SDN from soil, and this bacterium is G
+, spherical or shaft-like, bacterium colony is orange red.This bacterium can carry out aerobic denitrification during take SODIUMNITRATE as nitrogenous source, is respectively Carbon and nitrogen sources with sodium acetate and ammonium sulfate and can carries out heterotrophic nitrification, can be take ethanamide as sole carbon source and nitrogenous source grow.(environmental science, 2009,30 (12)) such as Chen Po ditchs between fields screen a strain heterotrophic nitrification-aerobic denitrification bacterium CPZ24 from livestock breeding wastewater.This bacterium is G
+, shaft-like, bacterium colony is orange red, is initially identified as to have a liking for pyridine rhodococcus (Rhodococuus pyridinivorans).In nitrifying process, this bacterium can all be removed ammonia nitrogen, nitrogen removal rate 98.70%; In aerobic denitrification, nitrate nitrogen clearance 66.74%, nitrogen removal rate 64.27%.The removal efficient of above-mentioned bacterial strains when nitrated and denitrification denitrogenation needs further to improve.
The screening of methanol degrading bacteria strain is that the biological treatment of trade effluent provides the foundation, and the methyl bacillus is the quasi-microorganism of methyl alcohol of can effectively degrading.Some methyl bacillus can affect nitrogen cycle by the conversion of ammonium salt and nitrate.CN200710175890.X discloses a strain recombination methyl nourishment bacillus MB202, produces Serine at the resting cell that utilizes recombination methyl nourishment bacillus MB202, and prospect all is widely used in processing methanol waste water.Madhaiyan etc. (Int J Syst Evol Microbiol, 2009,59 (Pt 1)) separate acquisition one strain methyl bacillus (Methylobacterium phyllosphaerae sp.nov.), this bacterium pinkiness from paddy rice.Chao Hongjun (microbiology circular, 2009,36 (11)) etc. isolates the bacterium that strain more than 200 can be take methyl alcohol as sole carbon source from methane tank residue, wherein a strain is initially identified as methyl bacillus (Methylobacterium sp.) MB200.Studies show that, this bacterium can utilize methyl alcohol, ethanol, Succinic Acid etc. as sole carbon source.(the agro-environment science journal such as Li Baoqing, 2007,26) make sole carbon source with methyl alcohol, utilize the method for enrichment culture from the active sludge of chemical plant, to be separated to a strain methanol degrading bacteria CYW-32, be accredited as and have a liking for organic methyl bacillus (Methylobacterium organophilium).But by the colorimetric acid system clear and definite the methyl alcohol of bacterial strain CYW-32 efficient degradation 500mg/L in 24h, degradation rate reaches 97.75%.Kong Qingsheng etc. (chemistry and biotechnology, 2009,26 (1)) screen a strain and can utilize methyl alcohol to be the bacterium SDM11 of sole carbon source and energy growth, this bacterium G from active sludge
-, rod-short is initially identified as the methylotrophy Pseudomonas.Be 0.6% at 30 ℃ of temperature, methanol content, under the optimum growing condition of pH7.0, the degradation rate of methyl alcohol reaches 82.5%.
Summary of the invention
For the deficiencies in the prior art, the invention provides the aerobic denitrification methyl bacillus that a strain has the spontaneous growth advantage, this bacterial strain has higher aerobic denitrification efficient, also have the performance that heterotrophic nitrification ammonia nitrogen removal and aerobic denitrification carry out simultaneously, nitrification and denitrification is combined in the process.
The invention provides a strain aerobic denitrification methyl bacillus, methyl bacillus (Methylobacterium phyllosphaerae) SDN-3, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 03 11st, 2010, deposit number is CGMCC No.3660.
Methyl bacillus SDN-3 of the present invention is Gram-negative bacteria, and thalline is shaft-like, can move; The bacterium colony circle is tiny, is orange, and neat in edge is smooth; Catalase is positive, and oxidase positive can be utilized several kinds of carbon source.Physio-biochemical characteristics are as shown in table 1.
The physio-biochemical characteristics of table 1 methyl bacillus SDN-3
Annotate: "+" expression positive reaction maybe can utilize; "-" expression negative reaction maybe can not be utilized.
The 16SrRNA gene sequencing analytical results of methyl bacillus SDN-3 of the present invention is seen sequence table.
The nitrate reductase activity of methyl bacillus SDN-3 of the present invention is high, all can express under aerobic and limited oxygen condition, has aerobic denitrification and limit oxygen denitrification function, also has the function of finishing simultaneously nitrification and denitrification.Specific performance comprises: (1) utilizes cheap carbon source (such as methyl alcohol or contain COD waste water etc.), and nitrate nitrogen carries out the aerobic denitrification denitrogenation as only nitrogen source; (2) utilize cheap carbon source (such as methyl alcohol or contain COD waste water etc.), ammonia nitrogen carries out the heterotrophic nitrification-aerobic denitrification simultaneous denitrification as only nitrogen source.
Methyl bacillus SDN-3 of the present invention is 22 ℃～40 ℃ of temperature, and biological activity is stable under the condition of pH7.0～9.5, and denitrification effect is good.
Methyl bacillus SDN-3 of the present invention is applied in sewage treatment area, but the organic carbon source of enduring high-concentration be applicable to process and contain COD, contain the waste water of nitrate nitrogen or ammonia nitrogen simultaneously, nitrogen removal rate is greater than 90%, the total nitrogen average removal rate is greater than 9mg/ (Lh); The COD clearance is greater than 85%.
Methyl bacillus SDN-3 of the present invention is applied to the aerobic denitrification process of sewage disposal, and its operational condition is: dissolved oxygen is 0.2mg/L～2.5mg/L, and carbon-nitrogen mass ratio is 3.0～6.0, and temperature is 22 ℃～40 ℃, pH7.0～9.5.
Methyl bacillus SDN-3 of the present invention is applied to the Simultaneous Nitrification denitrification process of sewage disposal, as being used for containing the Simultaneous Nitrification denitrification treating processes of ammonia-nitrogen sewage, its operational condition is: dissolved oxygen is 0.5mg/L～5.0mg/L, carbon-nitrogen mass ratio is 3.0～6.0,22 ℃～40 ℃ of temperature, pH7.0～9.5.
Methyl bacillus SDN-3 of the present invention in the situation that the undesirable bioremediation agents that can be used as of sewage water denitrification treatment effect add.
Methyl bacillus SDN-3 of the present invention can directly apply to the denitrification denitrogenation that contains nitrate nitrogen waste water, also can be applied to the denitrogenation of nitrogen-containing wastewater, is the bacterium that a strain has heterotrophic nitrification-aerobic denitrification capability.Can shorten the start time of system after this bacterium is come into operation, in all kinds of denitrogenation of waste water treating processess, have boundless application prospect, be particularly suitable in the synchronous nitration and denitrification biological denitrification process process.
Methyl bacillus SDN-3 of the present invention is the nitrification bacteria with aerobic denitrification characteristic, make nitrification and denitrification become possibility a unit inter-sync, overcome simultaneously the contradiction of conventional processes aspect processing efficiency and economic and practical two, realized waste water high-efficiency and economic denitrogenation.Therefore, obtain efficient degradation bacteria strains, and make it become the dominant population in the reaction system and bring into play the fast denitrogenation effect, will accelerate the process of synchronous nitration and denitrification technique industrial applications.
Biomaterial preservation explanation
Methyl bacillus provided by the invention (Methylobacterium phyllosphaerae) SDN-3 bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC); Address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Deposit number: CGMCC No.3660; Preservation date: on 03 11st, 2010.
Description of drawings
Fig. 1 is that methyl bacillus SDN-3 of the present invention processes the total nitrogen removal effect that contains different carbon source waste water.
Embodiment
Below specify separation screening process and the application example of bacterial strain of the present invention, the percentage composition that does not wherein indicate benchmark is weight percentage.
Bacterial strain of the present invention adopts dilution-plate method separation screening from the active sludge flora to have the bacterial strain of aerobic denitrification capability, and active sludge is from sewage work of Fushun No.3 Petroleum Factory.
Separating screening method is as follows:
1. adopt dilution-plate method: get a little active sludge, coat primary dcreening operation solid medium dull and stereotyped (methyl alcohol 5.0mL, KNO behind the gradient dilution
31.0g, KH
2PO
41.0g, FeCl
2H
2O 0.5g, CaCl
2H
2O 0.2g, MgSO
41.0g, BTB 1.0mL, water 1.0L, pH7.0～7.5, the ratio in 2% adds agar), 30 ℃～35 ℃ cultivations are selected substratum to become blue bacterial strain and are carried out purifying.Through repeatedly switching, screening obtains 3 strain aerobic denitrifying bacterias, is numbered SDN-1～SDN-3.
2. the aerobic denitrification nitrogen removal performance relatively: will screen the 3 strain bacterium that obtain and be inoculated in multiple sieve liquid nutrient medium (methyl alcohol 5.0mL, KNO
31.0g, KH
2PO
41.0g, FeCl
2H
2O 0.5g, CaCl
2H
2O 0.2g, MgSO
41.0g, water 1.0L, pH7.0～7.5), 30 ℃～35 ℃ cultivations.Obtain nitrate nitrogen and nitrogen removal rate all greater than 91% bacterial strain SDN-3, other bacterial strain nitrogen removal rate is 60%～70%.Aerobic denitrification capability through repeatedly experiment showed, bacterial strain SDN-3 can genetic stability.
3. the preparation of seed liquor: the bacterial strain SDN-3 access 150mL that purifying is cultivated sieves liquid nutrient medium again, cultivates 3 days for 30 ℃～35 ℃.The biomass of subsequent wastewater treatment all adds according to the amount of the centrifugal rear 10g/L of seed culture fluid.
4. the utilization of carbon source ability is investigated: select screening aerobic denitrifying bacteria Soduxin, l-asparagine, Trisodium Citrate, sodium acetate, ethanol and methyl alcohol etc. comparatively commonly used to investigate for carbon source.The result shows that bacterial strain SDN-3 can utilize several kinds of carbon source, especially carries out aerobic denitrification take methyl alcohol as carbon source, and nitrogen removal rate reaches 90%, COD clearance and reaches 97%.SDN-3 processes the total nitrogen removal effect that contains different carbon source waste water and sees accompanying drawing 1.
5. the heterotrophic nitrification ability is investigated: take ammonia nitrogen as nitrogenous source, methyl alcohol is that carbon source is carried out the investigation of heterotrophic nitrification-aerobic denitrification ability.The result shows that SDN-3 has stronger heterotrophic nitrification-aerobic denitrification ability, and ammonia nitrogen removal frank is greater than 95%, nitrogen removal rate greater than 90%, COD clearance greater than 98%.
Embodiment 1
The detection of the aerobic denitrification capability of methyl bacillus SDN-3.
Preparation simulated wastewater I, take nitrate nitrogen as only nitrogen source, starting point concentration is 277mg/L, carbon-nitrogen mass ratio is 5.3.Be 0.5mg/L at dissolved oxygen (DO), 24 ℃ of temperature are processed 24h under the condition of pH7.0.The nitrate nitrogen clearance is greater than 95%; Nitrogen removal rate is greater than 90%, total nitrogen average removal rate 10.4mg/ (Lh); The COD clearance is greater than 85%.
Simulated wastewater I: methyl alcohol 5.0mL, KNO
32.0g, KH
2PO
41.0g, FeCl
2H
2O 0.5g, CaCl
2H
2O 0.2g, MgSO
41.0g, water 1.0L, pH7.0～7.5.
Embodiment 2
The detection of the heterotrophic nitrification-aerobic denitrification capability of methyl bacillus SDN-3.
Preparation simulated wastewater II, take ammonia nitrogen as only nitrogen source, starting point concentration 265mg/L, carbon-nitrogen mass ratio are 3.4.Be 2.5mg/L at DO, 35 ℃ of temperature are processed 26h under the condition of pH8.5.Ammonia nitrogen removal frank and nitrogen removal rate be all greater than 90%, total nitrogen average removal rate 9.2mg/ (Lh); The COD clearance is greater than 99%.
Simulated wastewater II: methyl alcohol 3.0mL, ammonium sulfate 1.25g, KH
2PO
41.0g, FeCl
2H
2O 0.5g, CaCl
2H
2O 0.2g, MgSO
41.0g, water 1.0L, pH7.0～7.5.
Embodiment 3
Methyl bacillus SDN-3 is applied to the gourmet powder waste water of high concentration COD.
Gourmet powder waste water water quality: ammonia nitrogen concentration is 300mg/L～450mg/L, and COD is 700mg/L～850mg/L, pH5.0～6.0.Utilize the nitrifier of this laboratory culture that mineralized nitrogen is nitrate nitrogen, nitrated water outlet is intake as denitrification, then utilizes SDN-3 to carry out the aerobic denitrification denitrogenation.Initial nitrate nitrogen concentration is 320mg/L, and the carbon source deficiency is added methyl alcohol, and making carbon-nitrogen mass ratio is 4.0.Be 1.5mg/L at DO, 30 ℃ of temperature, process 30h under the condition of pH7.0～8.5 after, the nitrate nitrogen clearance is greater than 90%; Nitrogen removal rate is greater than 85%, total nitrogen average removal rate 9.07mg/ (Lh); The COD clearance is greater than 75%.
Embodiment 4
Methyl bacillus SDN-3 is applied to the urea ammonia-containing water.
Urea waste water water quality: ammonia nitrogen concentration 450mg/L, pH9.0～9.8.
Be nitrate nitrogen without nitrifier with mineralized nitrogen, directly utilize SDN-3 to carry out the heterotrophic nitrification-aerobic denitrification denitrogenation.The carbon source deficiency is added methyl alcohol, and making carbon-nitrogen mass ratio is 3.5.Be 2.5mg/L at DO, 30 ℃ of temperature, process 42h under the condition of pH7.0～8.5 after, ammonia nitrogen removal frank is greater than 95%; Nitrogen removal rate is greater than 90%, total nitrogen average removal rate 9.64mg/ (Lh); The COD clearance is greater than 96%.
Claims (5)
1. a strain aerobic denitrification methyl bacillus, methyl bacillus (Methylobacterium phyllosphaerae) SDN-3, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 03 11st, 2010, deposit number is CGMCC No.3660.
2. the application of the described methyl bacillus of claim 1 SDN-3 in sewage treatment area, sewage are to contain COD, contain the waste water of nitrate nitrogen or ammonia nitrogen simultaneously.
3. according to application claimed in claim 2, it is characterized in that: methyl bacillus SDN-3 is applicable to process and contains COD, contains the waste water of nitrate nitrogen or ammonia nitrogen simultaneously, and nitrogen removal rate is greater than 90%, and the total nitrogen average removal rate is greater than 9mg/ (Lh); The COD clearance is greater than 85%.
4. the application of the described methyl bacillus of claim 1 SDN-3 in the aerobic denitrification process that sewage water denitrification is processed, its operational condition is: dissolved oxygen is 0.2mg/L～2.5mg/L, carbon-nitrogen mass ratio is 3.0～6.0,22 ℃～40 ℃ of temperature, pH7.0～9.5.
5. the described methyl bacillus of claim 1 SDN-3 is in the Simultaneous Nitrification of sewage water denitrification processing and the application in the denitrification process, its operational condition is: dissolved oxygen is 0.5mg/L～5.0mg/L, carbon-nitrogen mass ratio is 3.0～6.0,22 ℃～40 ℃ of temperature, pH7.0～9.5.
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