CN101921708A - High-density culture method of mixed denitrification microorganism flora - Google Patents
High-density culture method of mixed denitrification microorganism flora Download PDFInfo
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- CN101921708A CN101921708A CN 201010244944 CN201010244944A CN101921708A CN 101921708 A CN101921708 A CN 101921708A CN 201010244944 CN201010244944 CN 201010244944 CN 201010244944 A CN201010244944 A CN 201010244944A CN 101921708 A CN101921708 A CN 101921708A
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Abstract
The invention provides a high-density culture method of mixed denitrification microorganism flora. The culture refers to the mixed culture of aerobic denitrification microorganism flora. The key points of the invention are as follows: keeping pH between 7.0 and 8.5 in the process of culture; starting to continuously feed glucose, ammonium sulfate and sodium succinate during the fifth hour to the tenth hour of the culture process; keeping concentration of glucose in the culture medium at 10-30g/L, concentration of ammonium sulfate at 5-25g/L, concentration of sodium succinate at 0.5-5g/L; and carrying out aerobic culture at 28-32 DEG C to obtain the mixed denitrification microorganism flora. In the invention, by continuously feeding ammonium sulfate and sodium succinate, the yield of the denitrification microorganism flora is remarkably improved, and the cell dry weight of the denitrification microorganism flora can reach 20g/L and above; and in addition, on the precondition of ensuring thallus weight, the pH value of phosphate buffer is kept at 8, the flora culture time is reduced from 48h to 24h, as a result, the culture efficiency is greatly improved.
Description
(1) technical field
The object of the invention provides a kind of high-density cultivation method of mixed denitrification microorganism flora.
(2) background technology
Along with the quick progress and the industrialized develop rapidly of science and technology, the people's standard of living improves day by day, but a large amount of simultaneously nitrogenous effluent that produces has brought a series of environmental problem.
Handle both at home and abroad at present the treatment process of nitrogenous effluent, mainly contain blow-off method, gas is put forward absorption method, chemical precipitation method, ion exchange method, electrochemical oxidation process and break point chlorination method etc.All there are some problems in the whole bag of tricks, and is not good such as treatment effect, complex process, operating mode be difficult to grasp and processing cost higher.By contrast, biological denitrificaion method economical and effective comparatively.Biological denitrifier is a kind of biological reinforced preparation that is specifically designed to out the middle ammonia nitrogen that anhydrates, and in order to reduce production costs, enhances productivity, and what this patent was breakthrough is applied to mixed denitrification microorganism with the high density fermentation technology, reaches good effect.
In physical environment or the biological treatment system, microorganism passes through the vital movement degradation of contaminant of self, objectionable impurities is decomposed into stablizes harmless small-molecule substance, as CO
2And H
2O.Along with industrial expansion, the discharging meeting of some hazardous and noxious substances produces certain toxic action to environmental microorganism, make and lack enough corresponding pollutents of microbiological deterioration in the biological degradation system, or the microorganism growth that suppresses to have the complicated pollutent performance of degraded, need a very long time microorganism to be adapted to gradually by modes such as the inducing of enzyme, transfer of genetic materials.The quantification technology of effective microbe is to introduce the microorganism with specific function in traditional biological treatment system, improve the concentration of effective microbe, enhancing is to the degradation capability of difficult degradation pollutent, improve its degradation rate, and improve the removal usefulness of original biological treatment system target contaminant.
At present the more traditional denitride technology that adopts has a technical bottleneck, and the nitrifying bacteria community rate of propagation is slow and be difficult to keep higher biological concentration, causes total hrt longer, and organic loading is lower, thereby has increased investment and running cost.And a little less than the bio-denitrification technology impact resistance, ammonia nitrogen in high density and nitrite water inlet can suppress the growth of nitrifier.
The Chinese invention patent of patent No. CN200510094737.5 provides a kind of high-density bacterium culturing method and biological reaction apparatus thereof, a kind of cell culture processes and biological reaction apparatus thereof have been described, relate in particular to a kind of cell high-density circulating or continous way cultural method and biological reaction apparatus thereof, it is characterized in that on bio-reactor, installing additional inorganic membrane filtration assembly and feed supplement jar, and the three is connected to a bioreactor system that can be used for cell cycle formula high-density culture or the cultivation of high-density continous way by certain control loop.But structure is too complicated, and crucial substratum difference, and its running technical process and be not suitable among the present invention cultivation at the mixed culture denitrification microorganism.
Number of patent application provides a kind of high-density cultivation method of lactic acid bacteria for kraut for the CN200610039750.5 Chinese patent application, mentioned that employing conventional plant milk-acid bacteria is a bacterial classification, through conventional method activation, enlarged culturing, be inoculated in the fermention medium of recipe configuration routinely, it is characterized in that be 6.3～6.6 with fermention medium with the buffer salt solution adjust pH that contains 0.2%～0.3% Trisodium Citrate, 0.2%～0.3% Sodium phosphate dibasic, 0.1%～0.2% SODIUM PHOSPHATE, MONOBASIC; In whole culturing process, stream adds ammoniacal liquor to keep the pH value stabilization 5.8～6.2; Bacterium liquid is added 500ml～700ml fresh culture after cultivating 20h; Bacterium liquid is centrifugal after cultivating and finishing, and adds the composite protectant of being made up of 10%～15% skimming milk, 5%～7% Sodium Glutamate, 2%～3% glycerine and 7%～8% maltose, vacuum lyophilization.Though also be to adopt high-density cultivation method, with every technical indicator of cultivation denitrification microorganism all by notable difference.
Mention high density fermentation in the pertinent literatures such as " high-density culture of mixed denitrification microorganism flora " (Liu Geng Jinju step on as etc.) and cultivated mixed denitrification microorganism, utilize the mixed culture technique of microorganism, studied nitrated-denitrifying biological denitrification process of while under the aerobic condition.The appropriate pH scope of mixed denitrification microorganism flora growth is 7～10, explored the pH control strategy of realizing the mixed denitrification microorganism flora high-density culture on the 5L fermentor tank: earlier fermentation is mended acid control pH≤8, the fermentation middle and later periods is not controlled the pH value, can shorten the growth cycle of thalline, improve the ammonia nitrogen degradation speed of thalline, the cell mass concentration reaches 3.9g/L, than having improved 62.5% under the natural pH condition.And on the 10L fermentor tank, do further to cultivate, verified the feasibility of its pH control strategy.By add (NH in batches
4)
2SO
4Make that bacterium is dense has further improved 15.4%, the final cell dry weight is 4.5g/L.But its cell concentration is still lower, does not far reach the requirement of high-concentration culturing.
(3) summary of the invention
The present invention seeks to a kind of high-density cultivation method of mixed denitrification microorganism flora, add ammonium sulfate, sodium succinate by Continuous Flow, and to keep phosphoric acid buffer pH value be 8, and the flora incubation time is after 24 hours, and the dry cell weight of the denitrogenation flora that obtains can reach 20g/L.
The technical solution used in the present invention is:
A kind of high-density cultivation method of mixed denitrification microorganism flora, described cultivation is the mixed culture of aerobic denitrification microorganism flora, main points of the present invention are: keeping pH in the culturing process is 7.0～8.5, and played (preferably since the 5th～7 hour) Continuous Flow on the 5th～10 hour and add glucose, ammonium sulfate and sodium succinate cultivating, keep that glucose concn is that 10～30g/L, ammonium sulfate concentrations are that 5～25g/L, sodium succinate concentration are 0.5～5g/L in the substratum, under 28～32 ℃, carry out aerobic cultivation and obtain mixed denitrification microorganism flora.
Fed batch cultivation (claiming feeding culture again) is the characteristics according to strain growth and initial medium, some stage of batch culture in some way intermittently or add fresh culture continuously, make the training method that production time of thalline and meta-bolites thereof prolongs.The feed supplement mode can be divided into feedback feed supplement and non-feedback feed supplement two classes: the feedback feed supplement comprises pH-stat method, DO-stat method, cell concentration back-to-back method, breathes commercial law etc., and non-feedback feed supplement can be divided into constant speed feeding culture, speed change feeding culture and index feeding culture.
Because nitrated microorganism produces acid in nitrifying process, when carrying out denitrification, denitrifying microorganism then produces alkali, and the suitableeest growth pH value of nitrated microorganism and denitrifying microorganism is also different, and mixed bacterial has powerful self-regulating function to the initial pH of certain limit in addition.Therefore, the pH in the culturing process must be controlled at the harmonic growth that zone of reasonableness could be realized mixed bacterial, keep higher biomass growth rate and ammonia nitrogen degradation speed jointly.
High-density culture is a relative concept, is that stem cell weighs/liter (DCW/L) in order to the unit of describing.In broad terms, every cell density is than higher, so that all can become high-density culture near the cultivation of its theoretical value.High-density culture has the concentration of thalline in the fermentation density that improves thalline or the unit volume nutrient solution, and then improves volume productivity; Dwindle the bio-reactor volume, reduce investment of production equipment; Strengthen downstream separation and extract, and reduce advantage such as wastewater flow rate.Its net effect reaches and improves specific production rate (output of product in unit volume, unit time), reduces production costs, and the commercialization process of expedite product improves the competitive power of product on market.
In the process of high-density fermentation, nutritional control is crucial, it is too high that carbon source, nitrogenous source and inorganic salt can cause solution osmotic pressure in the fermention medium, cell dehydration death, tend to suppress the growth of thalline, the purpose efficiency of pcr product is descended, and excessive carbon source can make the cell ramp, cause dissolved oxygen sharply to descend.High-density culture adopts feed supplement to cultivate usually, makes various medium components be lower than inhibition concentration.
Broad sense, every cell density so that all can be described as high-density culture near the cultivation of its theoretical value, it is generally acknowledged that its higher limit is 150～200g (DCW/L) than higher, and lower value is 20～30g (DCW/L).Because Nitromonas among the present invention and Nitrosomas are as the bacterium more slowly that grows, therefore can reach 20g/L at 48 hours dry cell weights can be defined as high density fermentation with it.
The substratum that is used to cultivate described aerobic denitrification microorganism flora can be configured by this area routine, form such as the substratum in " high-density culture of mixed denitrification microorganism flora ", preferably, substratum starting point concentration of the present invention is composed as follows: NaHCO
31.0～5.0g/L, sodium succinate 0.5～2.0g/L, (NH
4)
2SO
40.5～2.0g/L, KH
2PO
40.01～0.15g/L, K
2HPO
40.5～2.0g/L, NaCl 0.1～1.0g/L, FeSO
40.1～1.0g/L, MgSO
40.05～0.5g/L, MnSO
40.01～0.2g/L, solvent are water, pH7.0～8.5.
More preferred, described substratum starting point concentration is composed as follows: NaHCO
31.85g/L, sodium succinate 0.954g/L, (NH
4)
2SO
41.0g/L, KH
2PO
40.06g/L, K
2HPO
41.0g/L, NaCl 0.5g/L, FeSO
40.4g/L, MgSO
40.25g/L, MnSO
40.08/L solvent is a water, pH8.0.
Preferably, described cultivation is carried out under pH8.0,30 ℃, and total incubation time is 24～48h.
Described aerobic denitrification microorganism flora can be the conventional flora in this area, such as nitrifier, denitrifying bacteria, nitrococcus etc., can screen acquisition according to a conventional method from soil or water, also can adopt commercial composite bacterium powder.Preferably, described aerobic denitrification microorganism flora is the mixing of nitrifier and nitrococcus, and for example the present patent application people is in the nitrifier CCTCC No:M 2010001 of screening acquisition in first to file CN201010107453.6 and the combination of nitrococcus CCTCC No:M 2010002.The blending ratio of nitrifier and nitrococcus can be unrestricted, in fact, because influencing each other and restricting between the different microorganisms flora, under the nitrifier of in Sewage treatment systems, initially dropping into and the uncertain situation of nitrococcus ratio, cultivate through domestication, two kinds of floras can reach certain balance in the final treatment system.Preferably, nitrifier and nitrococcus initial inoculation thalline ratio are 0.5～2: 1.
Preferably, described cultivation rose in the 5th hour the beginning Continuous Flow add the beginning Continuous Flow add glucose, ammonium sulfate and sodium succinate, keep that glucose concn is that 20g/L, ammonium sulfate concentrations are that 15g/L, sodium succinate concentration are 2.2g/L in the substratum, under 30 ℃, carry out aerobic cultivation, total incubation time 24h can obtain described mixed denitrification microorganism flora.
Beneficial effect of the present invention is mainly reflected in: the present invention adds ammonium sulfate, sodium succinate by Continuous Flow, makes denitrogenation flora yield significantly improve, the dry cell weight of denitrogenation flora can reach 20g/L and more than; Under the prerequisite that guarantees the thalline quality, keeping phosphoric acid buffer pH is 8, and the flora incubation time was shortened to 24 hours from 48 hours, has improved efficient greatly.
(4) description of drawings
Fig. 1 is embodiment 1 a mixed bacterium growth curve chart;
Fig. 2 is a mixed bacterium growth curve chart under the embodiment 2 different pH values.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1:
The substratum starting point concentration is composed as follows: NaHCO
31.85g/L, sodium succinate 0.954g/L, (NH
4)
2SO
41.0g/L, KH
2PO
40.06g/L, K
2HPO
41.0g/L, NaCl 0.5g/L, FeSO
40.4g/L, MgSO
40.25g/L, MnSO
40.08/L solvent is a water, pH8.0.
(the prosperous generation thing in Cangzhou technical institute produces for above-mentioned commercial nitrifier of substratum 250mL/ bottle graft kind and denitrifying bacteria composite bacterium powder 10g, total viable count 〉=8,000,000,000/gram), one bottle was played stream on the 10th hour in the cultivation beginning and adds glucose, ammonium sulfate and sodium succinate, making and keeping glucose concn in the substratum is that 20g/L, ammonium sulfate concentrations are that 15g/L, sodium succinate concentration are 2.2g/L, 30 ℃, 250r/min, the following 48h that cultivates of air flow 1vvm air conditions; Do not add any material in another flask culture process, cultivate under the same conditions, record the mixed bacterium growth curve as shown in Figure 1.
As shown in Figure 1, mixed bacterial is not when stream adds any nutritive substance, only can reach the dry cell weight of 13g/L behind the 48h, and after continuous feeding kept 2% glucose, 1.5% ammonium sulfate, 0.22% sodium succinate (not controlling the pH value in the culturing process), cell concentration can reach 20g/L behind 48h.
Embodiment 2:
The substratum starting point concentration is composed as follows: NaHCO
32.0g/L, sodium succinate 0.8g/L, (NH
4)
2SO
41.2g/L, KH
2PO
40.05g/L, K
2HPO
41.2g/L, NaCl 0.8g/L, FeSO
40.5g/L, MgSO
40.1g/L, MnSO
40.1/L solvent is a water, pH7.0～9.0.
Mixed bacteria liquid 12g (the Hebei beneficial microorganism technology company limited of the commercial nitrobacteria of substratum 250mL/ bottle graft kind, nitrite bacteria and the denitrifying bacterium of above-mentioned different pH value (pH6.0, pH7.0, pH8.0, pH9.0), model XH01), play current-sharing on the 5th hour in the cultivation beginning and add glucose, ammonium sulfate and sodium succinate, making and keeping glucose concn in the substratum is that 18g/L, ammonium sulfate concentrations are that 16g/L, sodium succinate concentration are 2.5g/L, 30 ℃, 250r/min, the following 24h that cultivates of air flow 1vvm air conditions obtain the mixed bacterium growth curve and see Fig. 2.
As shown in Figure 2, pH is 8 o'clock, and the clean growth velocity of thalline only just can reach the effect of 18.5g/L when the 24h at incubation time.
Embodiment 3:
The substratum starting point concentration is composed as follows: NaHCO
31.85g/L, sodium succinate 0.954g/L, (NH
4)
2SO
41.0g/L, KH
2PO
40.06g/L, K
2HPO
41.0g/L, NaCl 0.5g/L, FeSO
40.4g/L, MgSO
40.25g/L, MnSO
40.08/L solvent is a water, pH8.0.
An amount of nitrifier CCTCC of above-mentioned culture medium inoculated No:M 2010001 and nitrococcus CCTCC No:M 2010002, the thalline initial proportion is 1: 1, culture condition: 30 ℃ of fermenting process controlled temperature, mixing speed are 350r/min, air flow is 1vvm.
Result parameter: the flora incubation time is 24 hours, and dry cell weight reaches 20.7g/L.
The sludge acclimatization process:
The microbial fermentation solution that above-mentioned fermentation is obtained adds biochemistry pool according to the dosage of 5% (v/v) (the microbial fermentation mixed solution that adds 5ml in the treatment sewage of every 100ml), keeps the active sludge after obtaining taming 7 days.
Embodiment 4:
Carry out making biological denitrifier after the high density fermentation mixed denitrification microorganism cultivates according to embodiment 3 schemes
Its technical process of this biological denitrifier is:
Biological denitrifier manufacture craft flow process: at first seed culture medium in shaking bottle carries out seed culture acquisition in 24 hours seed liquor with Nitromonas and Nitrosomas combined inoculation; Seed liquor is with 10% volume ratio inoculation fermentation substratum, cultivate in the beginning in the 5th hour stream add glucose, ammonium sulfate and and sodium succinate, making and keeping glucose concn in the substratum is that 20g/L, ammonium sulfate concentrations are that 15g/L, sodium succinate concentration are 2.2g/L, cultivates 24h under 30 ℃, 250r/min, blowing air amount 1vvm condition.Centrifugal collection thalline, the final water content that then thalline is diluted to solid preparation is controlled at about 8%, add an amount of skimming milk and make protective material, even spraying on the wheat bran carrier, airtight naturally preservation or refrigeration.
This biological denitrifier detects through the third party, obtains a result as following table:
| Test item | Detected result |
| Outward appearance | Pale brown toner end |
| The product total number of bacterial colony | 7,200,000,000 cfu/g |
| Ammonia nitrogen (waste water) | 69.4mg/L |
| Ammonia nitrogen (waste water adds biological denitrifier) | 43.2mg/L |
Embodiment 5:
Yanjing Bear (Zhejiangxiandu) Co., Ltd. uses high density fermentation mixed denitrification microorganism of the present invention, can keep stably reaching standard.Raw water quality is BOD
5(biological oxygen demand (BOD) BiologyOxygen Demmand on the five) 155mg/L, COD 310mg/L, total nitrogen 33mg/L, ammonia nitrogen 13mg/L respectively gets 1000ml, place Glass Containers, carry out the aerobic stage processing with this sewage works mud and by the mud after the domestication of embodiment 3 methods respectively, aeration rate is 1vvm.Behind the reaction 18h, the water sample water quality BOD of raw waste water station sludge treatment
515mg/L, COD 20mg/L, total nitrogen 16mg/L, ammonia nitrogen 5mg/L; The water sample water quality of utilizing acclimation sludge of the present invention to handle is BOD
550mg/L, COD 17mg/L, total nitrogen 4mg/L, ammonia nitrogen 2mg/L; Therefore adopt the mix bacterium agent of the technology of the present invention fermentation, overall efficiency can improve about 50%.
Embodiment 6:
Shaoxing sewage disposal Development Co., Ltd, use the art of this patent high density fermentation mixed denitrification microorganism, COD (chemical oxygen demand, chemical oxygen demand) 400mg/L, total nitrogen 30mg/L, ammonia nitrogen 10mg/L respectively gets 1000ml, places Glass Containers, carry out the aerobic stage processing with the said firm's sewage works mud and by the mud after the domestication of embodiment 3 methods respectively, aeration rate is 1vvm.Behind the reaction 18h, the water sample water quality BOD of raw waste water station sludge treatment
520mg/L, COD 40mg/L, total nitrogen 20mg/L, ammonia nitrogen 6mg/L; The water sample water quality of utilizing this patent acclimation sludge to handle is BOD
56mg/L, COD 20mg/L, total nitrogen 7mg/L, ammonia nitrogen 3mg/L; Therefore adopt the mix bacterium agent of the technology of the present invention high density fermentation, overall efficiency can improve about 50%.
Claims (6)
1. the high-density cultivation method of a mixed denitrification microorganism flora, described cultivation is the mixed culture of aerobic denitrification microorganism flora, it is characterized in that: keeping pH in the described culturing process is 7.0～8.5, and played the beginning Continuous Flow on the 5th～10 hour and add glucose, ammonium sulfate and sodium succinate cultivating, keep that glucose concn is that 10～30g/L, ammonium sulfate concentrations are that 5～25g/L, sodium succinate concentration are 0.5～5g/L in the substratum, under 28～32 ℃, carry out aerobic cultivation and obtain mixed denitrification microorganism flora.
2. the method for claim 1 is characterized in that described substratum starting point concentration is composed as follows: NaHCO
31.0～5.0g/L, sodium succinate 0.5～2.0g/L, (NH
4)
2SO
40.5～2.0g/L, KH
2PO
40.01～0.15g/L, K
2HPO
40.5～2.0g/L, NaCl 0.1～1.0g/L, FeSO
40.1～1.0g/L, MgSO
40.05～0.5g/L, MnSO
40.01～0.2g/L, solvent are water, pH7.0～8.5.
3. method as claimed in claim 2 is characterized in that described substratum starting point concentration is composed as follows: NaHCO
31.85g/L, sodium succinate 0.954g/L, (NH
4)
2SO
41.0g/L, KH
2PO
40.06g/L, K
2HPO
41.0g/L, NaCl 0.5g/L, FeSO
40.4g/L, MgSO
40.25g/L, MnSO
40.08/L solvent is a water, pH8.0.
4. the method for claim 1 is characterized in that described cultivation carries out under pH8.0,30 ℃, total incubation time is 24～48h.
5. the method for claim 1 is characterized in that described aerobic denitrification microorganism flora is the mixing of nitrifier and nitrococcus.
6. the method for claim 1, it is characterized in that described cultivation played the beginning Continuous Flow on the 5th hour and adds glucose, ammonium sulfate and sodium succinate, keep that glucose concn is that 20g/L, ammonium sulfate concentrations are that 15g/L, sodium succinate concentration are 2.2g/L in the substratum, under 30 ℃, carry out aerobic cultivation, total incubation time 24h can obtain described mixed denitrification microorganism flora.
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|---|---|---|---|---|
| CN105543093A (en) * | 2015-12-22 | 2016-05-04 | 天津凯英科技发展有限公司 | A preservative for an ammonia nitrogen degrading microbial inoculum and a preserving method thereof |
| CN106434412A (en) * | 2016-06-27 | 2017-02-22 | 郭洪伟 | Nitrobacterium continuous production method and production equipment thereof |
| CN109399876A (en) * | 2017-08-16 | 2019-03-01 | 天津大学 | A kind of acclimation method with humic acid reducing power activated sludge |
| CN111099740A (en) * | 2018-10-26 | 2020-05-05 | 中国石油化工股份有限公司 | Feed supplement control method for chemoautotrophic microorganism culture process |
| CN112746030A (en) * | 2019-10-30 | 2021-05-04 | 中国石油化工股份有限公司 | Culture method of nitrifying bacteria |
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2010
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105543093A (en) * | 2015-12-22 | 2016-05-04 | 天津凯英科技发展有限公司 | A preservative for an ammonia nitrogen degrading microbial inoculum and a preserving method thereof |
| CN105543093B (en) * | 2015-12-22 | 2019-10-01 | 天津凯英科技发展股份有限公司 | A kind of ammonia nitrogen degradation microbial inoculum preservative agent and store method |
| CN106434412A (en) * | 2016-06-27 | 2017-02-22 | 郭洪伟 | Nitrobacterium continuous production method and production equipment thereof |
| CN106434412B (en) * | 2016-06-27 | 2019-06-14 | 郭洪伟 | A kind of nitrobacteria continuous producing method and its production equipment |
| CN109399876A (en) * | 2017-08-16 | 2019-03-01 | 天津大学 | A kind of acclimation method with humic acid reducing power activated sludge |
| CN109399876B (en) * | 2017-08-16 | 2023-09-15 | 天津大学 | Domestication method of activated sludge with humic acid reducing capability |
| CN111099740A (en) * | 2018-10-26 | 2020-05-05 | 中国石油化工股份有限公司 | Feed supplement control method for chemoautotrophic microorganism culture process |
| CN112746030A (en) * | 2019-10-30 | 2021-05-04 | 中国石油化工股份有限公司 | Culture method of nitrifying bacteria |
| CN112746030B (en) * | 2019-10-30 | 2023-02-03 | 中国石油化工股份有限公司 | Culture method of nitrifying bacteria |
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| CN101921708B (en) | 2012-04-25 |
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