CN1594591A - Method for screening aerobic denitrifying bacteria - Google Patents
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- CN1594591A CN1594591A CN 200410043702 CN200410043702A CN1594591A CN 1594591 A CN1594591 A CN 1594591A CN 200410043702 CN200410043702 CN 200410043702 CN 200410043702 A CN200410043702 A CN 200410043702A CN 1594591 A CN1594591 A CN 1594591A
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Abstract
The invention relates to a high efficiency screening method for aerobic denitrifying bacteria. The invention includes the following steps: a. enriching bacteria employing domesticating anaerobic denitrifying sludge, b. separating and purifying domesticated bacteria from sludge using plate dilution method and plate streak method, c. preliminary screening for pure bacteria after separation and purification, obtaining bacteria with 50% TN removal rate, d. screening bacteria again, obtaining aerobic denitrifying bacteria. Accomplishment of aerobic denitrifying technology can not only overcome the shortcomings of traditional biological denitrification technology, but possess the following advantages: (1) can accomplishing denitrification, COD removal in same reactor, (2) shortening retention time under feedback regulation, (3) diminishing aeration volume, (4)alkaline denitrification produced can neutralizing with acid produced in nitration helping to realizing short range synchronous nitration and denitrification.
Description
Technical field:
The present invention relates to a kind of method of efficient screening aerobic denitrifying bacteria.
Background technology:
It is generally acknowledged that the denitrifying bacterium that carries out nitrate respiration all is an amphimicrobe,, just can induce needed nitrate reductase A of denitrification and nitrite reductase because only under oxygen free condition.Yet people constantly found the loss of total nitrogen under the aerobic condition in recent years, therefore, the research of aerobic denitrification were caused gradually investigator's attention.At present, the reason that the aerobic denitrification phenomenon is formed is final conclusion not still, and all from making local anaerobism or the microenvironment anaerobism is studied the aerobic denitrification technology, its operation control is complicated, system's fluctuation of service for most researchers both at home and abroad.Nitrogen in the water body can cause body eutrophication, so the removal of nitrogen has caused investigators' common concern.Yet, the traditional biological denitride technology, because of nitrifier and denitrifying bacteria are completely contradicted to the demand of oxygen, and it is unified that two processes of nitration denitrification are difficult on time and space, and have following drawback: (1) construction costs height, operation control is complicated; (2) acidity that produces in the nitrifying process need add the alkali neutralization, has not only increased processing costs, but also may cause secondary pollution; (3) system's impact resistance is poor, high density NH
4 +, NO
2 -The capital suppresses nitrobacteria growth.
Summary of the invention:
The objective of the invention is to adopt the method for screening aerobic denitrifying bacteria, realize the aerobic denitrification phenomenon from the biology angle, this technology is to realize the prerequisite of short distance synchronous nitration and denitrification, and contrast traditional biological denitride technology has absolute predominance.The present invention carries out according to following step: a, aerobic denitrifying bacteria enrichment: the method enriched biological flora of taking to tame anaerobic denitrification mud; B, bacterium separation and purification: the biological flora in the active sludge that will obtain after will taming adopts plate dilution method to separate and purifying with plate streak; C, aerobic denitrifying bacteria primary dcreening operation: the pure bacterium that separates, obtain behind the purifying is carried out primary dcreening operation, obtain the TN clearance is reached bacterial strain more than 50%; D, aerobic denitrifying bacteria sieve again: the bacterial strain behind the primary dcreening operation is carried out multiple sieve, obtain aerobic denitrifying bacteria.The realization of aerobic denitrification technology of the present invention can not only overcome the drawback of traditional biological denitride technology, also has following advantage: (1) can realize denitrogenation in the same reactor, remove COD; (2) under the adjusting of retroactive effect, shorten the residence time; (3) reduce aeration rate; (4) basicity that produces of denitrification process can in and the acidity that produces of nitrifying process, reduce basicity consumption; (5) help to realize the short distance synchronous nitration and denitrification.The above characteristics of aerobic denitrification technology can reduce the sewage disposal expense greatly, improve processing power and efficient.
Description of drawings:
Fig. 1 is aerobic denitrification bacterium concentrating device figure, and Fig. 2 is the multiple screen device figure of aerobic denitrifying bacteria.
Embodiment:
Embodiment one: present embodiment is carried out according to following step: a, aerobic denitrifying bacteria enrichment: the method enriched biological flora of taking to tame anaerobic denitrification mud, microorganism and environment, interaction between microorganism and microorganism is complicated delicate, flip-flop envrionment conditions (from anaerobic/anoxic → aerobic) does not only have the effect of enrichment, also can destroy the original eubiosis, to original flora also is a kind of destruction, so take to tame the means (the domestication device as shown in Figure 1) of anaerobic denitrification mud, change envrionment conditions gradually, promptly increase aeration rate gradually, aeration time, water inlet nitrate and nitrite concentration, reach the envrionment conditions that to screen bacterial strain, give the process of an adaptation of biological flora, finally make purpose flora-aerobic denitrifying bacteria become dominant microflora, reach the purpose of enrichment; B, bacterium separation and purification: the biological flora in the active sludge that will obtain after will taming adopts plate dilution method to separate and purifying with plate streak; C, aerobic denitrifying bacteria primary dcreening operation: the pure bacterium that separates, obtain behind the purifying is carried out primary dcreening operation, obtain the TN clearance is reached bacterial strain more than 50%; D, aerobic denitrifying bacteria sieve again: the bacterial strain behind the primary dcreening operation is carried out multiple sieve, obtain aerobic denitrifying bacteria.The method of described domestication anaerobic denitrification mud is achieved in that (1) handles high-concentration ammonia nitrogenous wastewater technology or the traditional anaerobic denitrification mud technology from the SBR method and gets active sludge; (2) domestication is initial, only with the FM water distribution as water inlet, every 2～3d, increase the content of DM water distribution nutrient solution in water distribution, up to water distribution all is the DM nutrient solution, tames to obtain after one month under the condition of DO value greater than 5mg/L, but the TN clearance of 500mg/L is reached active sludge more than 60%; Described FM water distribution is by 1.0g/L extractum carnis, 5.0g/L peptone and 1.0g/LKNO
3Form; Described DM water distribution is by 7.9g/LNa
2HPO
47H
2O, 1.5g/LKH
2PO
4, 0.3g/LNH
4Cl, 0.1g/LMgSO
47H
2O, 4.7g/L sodium succinate, 2ml trace element solution and oxidation state nitrogen are formed; Described oxidation state nitrogen is KNO
3And/or NaNO
2Described trace element solution is by 50.0g/L EDTA, 2.2g/L ZnSO
4, 5.5g/LCaCl
2, 5.06g/L MnCl
24H
2O, 5.0g/L FeSO
47H
2O, 1.1g/L (NH
4)
6Mo
7O
24H
2O, 1.57g/L CuSO
45H
2O and 1.61g/L CoCl
26 H
2O forms; The chemical oxygen demand (COD) of described water inlet (COD) is controlled at 4500～5500mg/L, and total nitrogen (TN) is at 450～550mg/L.The method of described aerobic denitrifying bacteria primary dcreening operation is achieved in that the pure bacterium inclined-plane that will obtain after the separation and purification, wash in the 250mL triangular flask that 100mL DM nutrient solution is housed with the DM nutrient solution after the sterilization, and in triangular flask, add the glass strain that bacterium is crossed in death of monks or nuns, bind up with gauze bottleneck with 9 layers, put into Air oscillator, in temperature is that 30 ℃, rotating speed are to cultivate 24h under the condition of 160rpm, obtains the TN clearance is reached bacterial strain more than 50%.The method that described aerobic denitrifying bacteria sieves again is achieved in that after (1) aerobic denitrifying bacteria is answered the 1000mLDM substratum of packing in the reactor of screen device (see figure 2) and seals, assemble with oxygen canister, open inlet mouth and venting port, with the flow aerating oxygen 3min of 3L/min; (2) close venting port, feed argon gas,, close inlet mouth, nutrient solution is cultivated when reactor pressure stops to feed argon gas during for malleation; (3) adopt NO in the nitrogen element track following assay method monitoring nutrient solution
3 --N, NO
2 -The variation of-N and DO.
Embodiment two: present embodiment is achieved in that
A, aerobic denitrifying bacteria enrichment: (1) is handled high-concentration ammonia nitrogenous wastewater technology or the traditional anaerobic denitrification mud technology from the SBR method and is got active sludge; (2) domestication is initial, only with the FM water distribution as water inlet, every 2～3d, increase the content of DM water distribution nutrient solution in water distribution, up to water distribution all is the DM nutrient solution, the COD of water inlet is controlled at 5000mg/L, total nitrogen (TN) is at 500mg/L, and in the domestication process, keep mud behind former dissolved oxygen (DO) concentration 2～3d, when changing water distribution, also DO is increased by 0.5～1mg/L, the purpose one of above way is that the nutrient environment of avoiding destroying biological flora suddenly damages flora, the 2nd, reach the suitable condition of aerobic denitrifying bacteria gradually, make such bacterium in the sludge acclimatization process, obtain enrichment, adopt the aforesaid method domestication can finish sludge acclimatization after one month, this moment, active sludge was by the initial aterrimus of domestication, diffusing shape changes the domestication light gray in latter stage into, cotton-shaped, be the aerobic sludge state.Domestication back active sludge is under the condition of DO value greater than 5mg/L, TN to 500mg/L can remove more than 60%, and the COD degradation rate can reach more than 80%, and possesses certain capacity of resisting impact load, sludge activity is strong, and has the aerobic denitrification flora of higher concentration.Take to tame the method enriched biological flora of anaerobic denitrification mud, microorganism and environment, interaction between microorganism and microorganism is complicated delicate, flip-flop envrionment conditions (from anaerobic/anoxic → aerobic) does not only have the effect of enrichment, also can destroy the original eubiosis, to original flora also is a kind of destruction, so take to tame the means of anaerobic denitrification mud, change envrionment conditions gradually, promptly increase aeration rate gradually, aeration time, water inlet nitrate and nitrite concentration, reach the envrionment conditions that to screen bacterial strain, give the process of an adaptation of biological flora, finally make purpose flora-aerobic denitrifying bacteria become dominant microflora, reach the purpose of enrichment.
B, bacterium separation and purification: the biological flora in the active sludge that will obtain after will taming adopts plate dilution method to separate and purifying with plate streak.
C, aerobic denitrifying bacteria primary dcreening operation: with the pure bacterium inclined-plane that obtains after the separation and purification, (the oxidation state nitrogen of adding is 1.0g/LKNO with the DM after the sterilization
3, 0.5g/LNaNO
2) nutrient solution washes in the 250mL triangular flask that 100mL DM nutrient solution is housed, and in each triangular flask, add the glass strains (as far as possible reducing of the influence of anaerobism microenvironment) of several the bacterium of going out to experimental result, bind up with gauze bottleneck (assurance aerobic condition) with 9 layers, put into Air oscillator, in temperature is that 30 ℃, rotating speed are to cultivate 24h under the condition of 160rpm, obtains the TN clearance is reached bacterial strain more than 50%.The bacterium liquid of getting before and after cultivating is measured TN concentration, investigates its clearance to TN.In this experiment, investigate to separate the 105 strain bacterium that the obtain clearance to TN respectively, wherein clearance has 21 strains at the bacterial strain more than 50%, and 40%～50% bacterial strain has 15 strains, and 30%～40% bacterial strain has 10 strains.It is generally acknowledged that the nitrogen that biological assimilation utilizes can not surpass 30%, so can think that this 46 strain bacterium has denitrification substantially under aerobic condition.In addition, the 17 strain bacterium that filter out from active sludge before the domestication are all lower to the TN clearance, the highest by only 17%.As seen, domestication back strain separated is significantly improved to the clearance of TN, and this result further confirms to tame the concentration that can improve the aerobic denitrification flora.But because assimilation has interference to the result, thus should take the method for multiple sieve further confirm the bacterial strain that screens whether be aerobic denitrifying bacteria.
D, aerobic denitrifying bacteria sieve again: the entire reaction device is airtight, reactor is made up of 2000mL tool plug airtight reagent bottle and pipe fitting, import and export two ends and be furnished with the bacterial filter that the aperture is 0.25 μ m, reactor is packed into and is sealed behind the 1000mLDM substratum, with the oxygen canister assembling, open inlet mouth and venting port, with the flow aerating oxygen 3min of 3L/min, the air of driving away in reactor and the pipeline removes nitrogen contained in the air, avoids the interference of nitrogen to measuring result.Close venting port, feed argon gas, determine that by the reading of gas-pressure meter reactor pressure is a malleation, stop to feed argon gas, close inlet mouth.Meanwhile, open magnetic stirring apparatus, purpose is to make the nutrient solution homogeneous and fully contact with top oxygen (to record DO in the culturing process and can maintain 8mg/L～10mg/L) all the time.The purpose that feeds argon gas is to make press strip spare is arranged: the one, make to form malleation in the reactor, and oxygen can be dissolved in the nutrient solution more fully, guarantee definitely aerobic; The 2nd, malleation can stop airborne nitrogen to enter reactor, makes the amount of the denitrifying product of the absolute representative of the amount that obtains the nitrogen in the gas sample.Thief hole 1 is monitored nitrogen growing amount (vapor-phase chromatography) in the culturing process in order to get the gas sample, and thief hole 2 is monitored NO in the nutrient solution in order to get the nutrient solution sample
3 --N, NO
2 -The variation of-N and DO etc.The substratum of experiment usefulness is the DM substratum, hydrogen acceptor KNO
30.8g/L other compositions are constant.In this experiment, 21 strains to primary dcreening operation have been done multiple sieve to the clearance of TN at the bacterial strain more than 50%, confirmed that they can carry out nitrate respiration under aerobic condition, denitrification percent can reach more than 95%, and the denitrification end products is a nitrogen, be that this 21 strain bacterium all is an aerobic denitrifying bacteria, the realization that this further proves aerobic denitrification in the acclimation sludge, be since mud inside efficiently, a large amount of aerobic denitrifying bacteria in action.
By above experimental result as can be known, adopting this method screening aerobic denitrifying bacteria is efficiently, and each step all plays an important role, and wherein sludge acclimatization work plays a key role, and the multiple sieve decisive role of aerobic denitrifying bacteria.
Claims (9)
1, the method for screening aerobic denitrifying bacteria is characterized in that it carries out according to following step: a, aerobic denitrifying bacteria enrichment: the method enriched biological flora of taking to tame anaerobic denitrification mud; B, bacterium separation and purification: the biological flora in the active sludge that will obtain after will taming adopts plate dilution method to separate and purifying with plate streak; C, aerobic denitrifying bacteria primary dcreening operation: the pure bacterium that separates, obtain behind the purifying is carried out primary dcreening operation, obtain the TN clearance is reached bacterial strain more than 50%; D, aerobic denitrifying bacteria sieve again: the bacterial strain behind the primary dcreening operation is carried out multiple sieve, obtain aerobic denitrifying bacteria.
2, the method for screening aerobic denitrifying bacteria according to claim 1, the method that it is characterized in that described domestication anaerobic denitrification mud are achieved in that (1) handles high-concentration ammonia nitrogenous wastewater technology or the traditional anaerobic denitrification mud technology from the SBR method and get active sludge; (2) domestication is initial, only with the FM water distribution as water inlet, every 2～3d, increase the content of DM water distribution nutrient solution in water distribution, up to water distribution all is the DM nutrient solution, tames to obtain after one month under the condition of DO value greater than 5mg/L, but the TN clearance of 500mg/L is reached active sludge more than 60%.
3, the method for screening aerobic denitrifying bacteria according to claim 2 is characterized in that described FM water distribution is by 1.0g/L extractum carnis, 5.0g/L peptone and 1.0g/LKNO
3Form.
4, the method for screening aerobic denitrifying bacteria according to claim 2 is characterized in that described DM water distribution is by 7.9g/L Na
2HPO
47H
2O, 1.5g/L KH
2PO
4, 0.3g/L NH
4Cl, 0.1g/LMgSO
47H
2O, 4.7g/L sodium succinate, 2ml trace element solution and oxidation state nitrogen are formed.
5, the method for screening aerobic denitrifying bacteria according to claim 4 is characterized in that described oxidation state nitrogen is KNO
3And/or NaNO
2
6, the method for screening aerobic denitrifying bacteria according to claim 4 is characterized in that described trace element solution is by 50.0g/L EDTA, 2.2g/L ZnSO
4, 5.5g/L CaCl
2, 5.06g/LMnCl
24H
2O, 5.0g/L FeSO
47H
2O, 1.1g/L (NH
4)
6Mo
7O
24H
2O, 1.57g/LCuSO
45H
2O and 1.61g/L CoCl
26H
2O forms.
7, the method for screening aerobic denitrifying bacteria according to claim 2 is characterized in that the COD of described water inlet is controlled at 4500～5500mg/L, and total nitrogen is controlled at 450～550mg/L.
8, the method for screening aerobic denitrifying bacteria according to claim 1, the method that it is characterized in that described aerobic denitrifying bacteria primary dcreening operation is achieved in that the pure bacterium inclined-plane that will obtain after the separation and purification, wash in the 250mL triangular flask that 100mL DM nutrient solution is housed with the DM nutrient solution after the sterilization, and in triangular flask, add the glass strain that bacterium is crossed in death of monks or nuns, bind up with gauze bottleneck with 9 layers, put into Air oscillator, in temperature is that 30 ℃, rotating speed are to cultivate 24h under the condition of 160rpm, obtains the TN clearance is reached bacterial strain more than 50%.
9, the method for screening aerobic denitrifying bacteria according to claim 1, it is characterized in that sealing after the method that described aerobic denitrifying bacteria sieves again is achieved in that the 1000mLDM substratum of packing in the reactor of the multiple screen device of (1) aerobic denitrifying bacteria, assemble with oxygen canister, open inlet mouth and venting port, with the flow aerating oxygen 3min of 3L/min; (2) close venting port, feed argon gas,, close inlet mouth, nutrient solution is cultivated when reactor pressure stops to feed argon gas during for malleation; (3) adopt NO in the nitrogen element track following assay method monitoring nutrient solution
3 --N, NO
2 -The variation of-N and DO.
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| CN100400650C (en) * | 2006-02-14 | 2008-07-09 | 浙江大学 | Separation and authentication method for Planctomyces with anaerobic ammoxidation activity |
| CN102443550A (en) * | 2010-10-12 | 2012-05-09 | 中国石油化工股份有限公司 | Screening method of denitrifying bacteria |
| CN101338282B (en) * | 2007-07-02 | 2012-05-23 | 中国科学院成都生物研究所 | Heterotrophic nitrification aerobic denitrifying bacteria, culturing method thereof and uses |
| CN102952765A (en) * | 2011-08-31 | 2013-03-06 | 中国石油化工股份有限公司 | Enrichment culture method of salt-tolerant efficient nitrosobacteria |
| CN105039225A (en) * | 2015-08-25 | 2015-11-11 | 哈尔滨工业大学 | Aerobic denitrifying bacterium strain and application thereof |
| CN105586290A (en) * | 2015-12-24 | 2016-05-18 | 中国科学院重庆绿色智能技术研究院 | Strain with heterotrophic nitrification and aerobic denitrification functions and application of strain |
| CN106399183A (en) * | 2016-10-19 | 2017-02-15 | 武夷学院 | Aerobic denitrification bacterium and application thereof |
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| CN110902820A (en) * | 2019-12-09 | 2020-03-24 | 深圳市水务(集团)有限公司 | Denitrifying bacterium domestication culture method |
| CN111100794A (en) * | 2019-12-27 | 2020-05-05 | 安徽国星生物化学有限公司 | Method for separating and screening high-efficiency denitrifying bacteria from sequencing batch pyridine wastewater activated sludge |
| CN111454859A (en) * | 2020-04-07 | 2020-07-28 | 山东海景天环保科技股份公司 | Method for cultivating low-temperature aerobic denitrifying bacteria |
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| CN100400650C (en) * | 2006-02-14 | 2008-07-09 | 浙江大学 | Separation and authentication method for Planctomyces with anaerobic ammoxidation activity |
| CN101338282B (en) * | 2007-07-02 | 2012-05-23 | 中国科学院成都生物研究所 | Heterotrophic nitrification aerobic denitrifying bacteria, culturing method thereof and uses |
| CN102443550A (en) * | 2010-10-12 | 2012-05-09 | 中国石油化工股份有限公司 | Screening method of denitrifying bacteria |
| CN102443550B (en) * | 2010-10-12 | 2014-05-21 | 中国石油化工股份有限公司 | Screening method of denitrifying bacteria |
| CN102952765A (en) * | 2011-08-31 | 2013-03-06 | 中国石油化工股份有限公司 | Enrichment culture method of salt-tolerant efficient nitrosobacteria |
| CN102952765B (en) * | 2011-08-31 | 2014-11-05 | 中国石油化工股份有限公司 | Enrichment culture method of salt-tolerant efficient nitrosobacteria |
| CN105039225A (en) * | 2015-08-25 | 2015-11-11 | 哈尔滨工业大学 | Aerobic denitrifying bacterium strain and application thereof |
| CN105039225B (en) * | 2015-08-25 | 2018-03-30 | 哈尔滨工业大学 | A kind of aerobic denitrifying bacteria and its application |
| CN105586290B (en) * | 2015-12-24 | 2019-07-23 | 中国科学院重庆绿色智能技术研究院 | A kind of bacterial strain and its application with heterotrophic nitrification and aerobic denitrification function |
| CN105586290A (en) * | 2015-12-24 | 2016-05-18 | 中国科学院重庆绿色智能技术研究院 | Strain with heterotrophic nitrification and aerobic denitrification functions and application of strain |
| CN106399183A (en) * | 2016-10-19 | 2017-02-15 | 武夷学院 | Aerobic denitrification bacterium and application thereof |
| CN109628309A (en) * | 2018-12-28 | 2019-04-16 | 江苏大学 | A kind of separation method of saline-alkali tolerant denitrifying microorganism bacterial strain |
| CN110902820A (en) * | 2019-12-09 | 2020-03-24 | 深圳市水务(集团)有限公司 | Denitrifying bacterium domestication culture method |
| CN111100794A (en) * | 2019-12-27 | 2020-05-05 | 安徽国星生物化学有限公司 | Method for separating and screening high-efficiency denitrifying bacteria from sequencing batch pyridine wastewater activated sludge |
| CN111454859A (en) * | 2020-04-07 | 2020-07-28 | 山东海景天环保科技股份公司 | Method for cultivating low-temperature aerobic denitrifying bacteria |
| CN115806909A (en) * | 2022-11-08 | 2023-03-17 | 哈尔滨工业大学（深圳） | Pseudomonas and separation method and application thereof |
| CN115806909B (en) * | 2022-11-08 | 2023-08-18 | 哈尔滨工业大学（深圳） | Pseudomonas and separation method and application thereof |
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