CN101921708B - High-density culture method of mixed denitrification microorganism flora - Google Patents
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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, mikrobe passes through the vital movement degradation of contaminant of self, objectionable impurities is decomposed into stablizes harmless small-molecule substance, like 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 mikrobe to be adapted to gradually through modes such as the inducing of enzyme, transfer of genetic materials.The quantification technology of effective microbe is in traditional biological treatment system, to introduce the mikrobe with specific function; 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.
The more traditional denitride technology that adopts at present 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 through certain loop.But structure is too complicated, and crucial substratum difference, and its running technical process and be not suitable among the present invention the cultivation to 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 one Chinese patent application; Mentioned that employing conventional plant milk-acid bacteria is a bacterial classification; Through conventional method activation, enlarged culturing; Be inoculated in fermention medium, it is characterized in that it is 6.3～6.6 that fermention medium is used 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 by the conventional formulation configuration; 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% SANMALT-S, vacuum lyophilization.Though also be to adopt high-density cultivation method, with each item 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 mikrobe, 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; On the 5L fermentor tank, explored the pH control strategy of realizing the mixed denitrification microorganism flora high-density culture: 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, improves 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.Through 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 through even flow; And to keep phosphoric acid buffer pH value be 8, and the flora incubation time is after 24 hours, and the dried cell weight of the denitrogenation flora that obtains can reach 20g/L.
The technical scheme that the present invention adopts is:
A kind of high-density cultivation method of mixed denitrification microorganism flora; Said 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) even 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 with certain mode intermittently or add fresh culture continuously, make the training method that PT of thalline and meta-bolites thereof prolongs.The feed supplement mode can be divided into two types of feedback feed supplement and non-feedback feed supplements: 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 mikrobe produces acid in nitrifying process; When carrying out denitrification, denitrifying microorganism then produces alkali; And the righttest growth pH value of nitrated mikrobe and denitrifying microorganism is also different, and mixed bacterial has powerful self-regulating function to the initial pH of certain limit in addition.Therefore, must the pH in the culturing process 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, and it is too high that carbon source, nitrogenous source and inorganic salt can cause solution osmotic pressure in the fermention medium, and cell dehydration is dead; 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 said 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 according to the invention is formed 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, said substratum starting point concentration is formed 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, said cultivation is carried out under pH8.0,30 ℃, and total incubation time is 24～48h.
Said aerobic denitrification microorganism flora can be the conventional flora in this area, such as nitrifier, denitrifying bacteria, nitrococcus etc., can from soil or water, obtain by the ordinary method screening, also can adopt commercial composite bacterium powder.Preferably; Said aerobic denitrification microorganism flora is the mixing of nitrifier and nitrococcus, and applicant for example of the present invention is in the combination of the nitrifier CCTCC No:M 2010001 and the nitrococcus CCTCC No:M 2010002 of screening acquisition in first to file CN201010107453.6.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 initial nitrifier of dropping into and the uncertain situation of nitrococcus ratio, through acclimation shaking culture, two kinds of floras can reach certain balance in the final treatment system in Sewage treatment systems.Preferably, nitrifier and nitrococcus initial inoculation thalline ratio are 0.5～2: 1.
Preferably; Said cultivation rose in the 5th hour the beginning even flow add the beginning even 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 said mixed denitrification microorganism flora.
Beneficial effect of the present invention is mainly reflected in: the present invention adds ammonium sulfate, sodium succinate through even flow, makes denitrogenation flora yield significantly improve, the dried 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
Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1:
The substratum starting point concentration is formed 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, and 30 ℃, 250r/min, air flow 1vvm air conditions are cultivated 48h down; Do not add any material in another flask culture process, cultivate under the same conditions, it is as shown in Figure 1 to record the mixed bacterium growth curve.
Can know by Fig. 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 formed 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.
The mixed bacteria liquid 12g 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) (Hebei beneficial microorganism technology ltd; 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, air flow 1vvm air conditions are cultivated 24h down, obtain the mixed bacterium growth curve and see Fig. 2.
Can be known that by Fig. 2 pH is at 8 o'clock, the clean growth velocity of thalline just can reach the effect of 18.5g/L when incubation time is merely 24h.
Embodiment 3:
The substratum starting point concentration is formed 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 dried 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 processing 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 Nitromonas and Nitrosomas combined inoculation seed culture medium in shaking bottle are carried out seed culture acquisition in 24 hours seed liquor; 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, under 30 ℃, 250r/min, blowing air amount 1vvm condition, cultivates 24h.Centrifugal collection thalline is controlled at the final water cut that thalline is diluted to solid preparation about 8% then, 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 follows:
| 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(BOD BiologyOxygen Demmand on the five) 155mg/L; COD 310mg/L; Total nitrogen 33mg/L, ammonia nitrogen 13mg/L respectively gets 1000ml, places 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 present invention's technology fermentation, overall efficiency can improve about 50%.
Embodiment 6:
Shaoxing WWT Development Co., Ltd uses patent art 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 technology high density fermentation of the present invention, overall efficiency can improve about 50%.
Claims (4)
1. the high-density cultivation method of a mixed denitrification microorganism flora; Said cultivation is the mixed culture of aerobic denitrification microorganism flora; It is characterized in that: keeping pH in the said culturing process is 7.0～8.5; And played the beginning even flow on the 5th～10 hour and add glucose, ammonium sulfate and sodium succinate cultivating; Keep that glucose concn is that 18～20g/L, ammonium sulfate concentrations are that 15～16g/L, sodium succinate concentration are 2.2～2.5g/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 said substratum starting point concentration forms 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. the method for claim 1 is characterized in that said cultivation carries out under pH8.0,30 ℃, total incubation time is 24～48h.
4. the method for claim 1; It is characterized in that said cultivation played the beginning even 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 said mixed denitrification microorganism flora.
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| 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 |
| CN106434412B (en) * | 2016-06-27 | 2019-06-14 | 郭洪伟 | A kind of nitrobacteria continuous producing method and its production equipment |
| CN109399876B (en) * | 2017-08-16 | 2023-09-15 | 天津大学 | Domestication method of activated sludge with humic acid reducing capability |
| CN111099740B (en) * | 2018-10-26 | 2022-06-07 | 中国石油化工股份有限公司 | Feed supplement control method for chemoautotrophic microorganism culture process |
| 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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| Lucija Foglar et al.High nitrate removal from synthetic wastewater with the mixed bacterial culture.《Bioresource Technology》.2004, * |
| 堵国成等.好氧同时硝化―反硝化脱氮微生物的混合培养.《食品与生物技术》.2002,(第05期), * |
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Effective date of registration: 20160928 Address after: 310012 , 164 Wen two road, Hangzhou, Zhejiang, Hangzhou Institute of Commerce Patentee after: ZHEJIANG SHUANGLIANG SHANGDA ENVIRONMENTAL PROTECTION Co.,Ltd. Address before: 310018, room 17-6, No. four, 302 Avenue, Hangzhou economic and Technological Development Zone, Hangzhou, Zhejiang Patentee before: ZHEJIANG SUNDAWATER Co.,Ltd. |
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| CP01 | Change in the name or title of a patent holder | ||
| CP01 | Change in the name or title of a patent holder |
Address after: Inside Hangzhou Business School, No.164 Wen'er Road, Xihu District, Hangzhou City, Zhejiang Province, 310012 Patentee after: Zhe JIANG Shuangliang Sunda Environment Protection Co.,Ltd. Address before: Inside Hangzhou Business School, No.164 Wen'er Road, Xihu District, Hangzhou City, Zhejiang Province, 310012 Patentee before: ZHEJIANG SHUANGLIANG SHANGDA ENVIRONMENTAL PROTECTION Co.,Ltd. |