CN112746052A - Culture method for improving resistance of nitrobacteria - Google Patents
Culture method for improving resistance of nitrobacteria Download PDFInfo
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- CN112746052A CN112746052A CN201911047179.5A CN201911047179A CN112746052A CN 112746052 A CN112746052 A CN 112746052A CN 201911047179 A CN201911047179 A CN 201911047179A CN 112746052 A CN112746052 A CN 112746052A
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- 241000108664 Nitrobacteria Species 0.000 title claims abstract description 32
- 238000012136 culture method Methods 0.000 title claims abstract description 11
- 241000894006 Bacteria Species 0.000 claims abstract description 49
- 238000005273 aeration Methods 0.000 claims abstract description 49
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 45
- 239000001301 oxygen Substances 0.000 claims abstract description 45
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 45
- 238000000034 method Methods 0.000 claims abstract description 40
- 230000001546 nitrifying effect Effects 0.000 claims abstract description 35
- 238000012258 culturing Methods 0.000 claims abstract description 6
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 claims description 27
- 244000005700 microbiome Species 0.000 claims description 17
- 239000001963 growth medium Substances 0.000 claims description 11
- 238000003756 stirring Methods 0.000 claims description 11
- 238000011081 inoculation Methods 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 3
- 238000013019 agitation Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 10
- 241001052560 Thallis Species 0.000 abstract description 8
- 230000001580 bacterial effect Effects 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 14
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 12
- 230000003647 oxidation Effects 0.000 description 7
- 238000007254 oxidation reaction Methods 0.000 description 7
- 229910021529 ammonia Inorganic materials 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 5
- 235000011130 ammonium sulphate Nutrition 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 4
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 4
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 4
- 239000001099 ammonium carbonate Substances 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 3
- 239000001110 calcium chloride Substances 0.000 description 3
- 229910001628 calcium chloride Inorganic materials 0.000 description 3
- 238000007599 discharging Methods 0.000 description 3
- 239000011790 ferrous sulphate Substances 0.000 description 3
- 235000003891 ferrous sulphate Nutrition 0.000 description 3
- 238000003306 harvesting Methods 0.000 description 3
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 3
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 3
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 3
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 3
- 238000009629 microbiological culture Methods 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 description 3
- 230000001590 oxidative effect Effects 0.000 description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 3
- 238000004062 sedimentation Methods 0.000 description 3
- 239000010865 sewage Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000001502 supplementing effect Effects 0.000 description 3
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 2
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241001453382 Nitrosomonadales Species 0.000 description 2
- CVTZKFWZDBJAHE-UHFFFAOYSA-N [N].N Chemical compound [N].N CVTZKFWZDBJAHE-UHFFFAOYSA-N 0.000 description 2
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 2
- 235000012501 ammonium carbonate Nutrition 0.000 description 2
- 235000019270 ammonium chloride Nutrition 0.000 description 2
- 150000003868 ammonium compounds Chemical class 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000589651 Zoogloea Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000001651 autotrophic effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- MWUXSHHQAYIFBG-UHFFFAOYSA-N nitrogen oxide Inorganic materials O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 1
- GQPLMRYTRLFLPF-UHFFFAOYSA-N nitrous oxide Inorganic materials [O-][N+]#N GQPLMRYTRLFLPF-UHFFFAOYSA-N 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
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Abstract
The invention discloses a culture method for improving resistance of nitrobacteria. The method comprises the following steps: (1) determining the upper limit value of the dissolved oxygen concentration in the culture system as DOmax and the corresponding aeration quantity Q; (2) determining the dissolved oxygen concentration of the nitrifying bacteria in the optimal growth state period and recording as DOtop; (3) in the process of culturing nitrobacteria, when the concentration of dissolved oxygen in a culture system is increased to 50-70% DOmax, and stays for 5-15 minutes, then a feeding system is started, and after the feeding system is started, the feeding system is stopped when the DO concentration is reduced by 20-40 percentage points; and circulating the steps until the culture is finished. The culture method can realize automatic control culture of the thalli, realize rapid growth and propagation, save production cost, and the obtained nitrobacteria has high activity and strong capability of resisting dissolved oxygen, thereby solving the problems of slow growth of the bacterial source and poor nitrification effect of high dissolved oxygen concentration under the condition of low load in industry.
Description
Technical Field
The invention belongs to the field of environmental microorganisms, and particularly relates to a culture method for improving resistance of nitrobacteria.
Background
The nitrifying bacteria mainly play a role in removing nitrogen-containing pollutants and comprise ammonia oxidizing bacteria and nitrite oxidizing bacteria. Wherein the ammonia oxidizing bacteria are responsible for oxidizing ammonia to nitrous acidSalt, nitrite-oxidizing bacteria are responsible for the further oxidation of nitrite to nitrate. Nitrifying bacteria belong to aerobic autotrophic microorganisms, and under the condition of sufficient nutrient supply, energy is obtained from the process of oxidizing ammonia and nitrite so as to assimilate CO2However, the nitrifying bacteria grow, so that the generation time of the nitrifying bacteria is long, the proliferation speed is slow, the industrial large-scale application is difficult, and in recent years, a large amount of research work is carried out on how to realize rapid culture of the nitrifying bacteria, and certain progress is achieved. Nitrifying bacteria are fragile and are easily influenced by various factors such as substrate concentration, Dissolved Oxygen (DO) concentration, pH, toxic substances and the like in the growth and application processes, particularly, some sewage treatment fields are excessively aerated in the final stage of reaction due to low operation load, the dissolved oxygen concentration is more than 5mg/L, on one hand, the nitrifying bacteria cannot tolerate the high-concentration dissolved oxygen environment, on the other hand, the excessively aerated dispersed zoogloea nitrifying bacteria are seriously lost along with water, and therefore, in order to improve the nitrification effect, although a method for adding a high-efficiency nitrifying bacteria agent is adopted in some sewage treatment fields, the nitrifying bacteria have poor resistance in the actual application process and DO not achieve the expected treatment effect.
The prior art adopts a batch feeding mode and a batch fed-batch mode, and can control the concentration of a substrate within a certain range, but cannot realize automatic control.
CN201611066275.0 provides an intermittent, ammonia nitrogen fed-batch operation alternate nitrobacteria flora screening and enrichment culture method, which mainly utilizes intermittent operation to gradually increase initial ammonia nitrogen load so as to increase ammonia nitrogen oxidation rate, and ammonia nitrogen fed-batch operation to maintain ammonia nitrogen oxidation rate and realize low nitrite accumulation rate. The method can realize the screening and enrichment culture of nitrobacteria flora in a short time, but the control process is relatively complex, and the automatic feeding culture can not be realized.
Therefore, how to realize efficient automatic control of batch culture of nitrobacteria is an important problem to be solved at present, and aiming at high-concentration dissolved oxygen environments such as sewage treatment plants operating under low load, a nitrobacteria capable of meeting the application of the specific application is also needed, and common nitrobacteria are difficult to adapt to the specific application.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a culture method for improving the resistance of nitrobacteria. The culture method can realize automatic control culture of the thalli, realize rapid growth and propagation, save production cost, and the obtained nitrobacteria has high activity and strong capability of resisting dissolved oxygen, thereby solving the problems of slow growth of the bacterial source and poor nitrification effect of high dissolved oxygen concentration under the condition of low load in industry.
The invention provides a culture method for improving resistance of nitrobacteria, which comprises the following steps:
(1) determining the upper limit value of the dissolved oxygen concentration in the culture system as DOmax and the corresponding aeration quantity Q;
(2) determining the dissolved oxygen concentration of the nitrifying bacteria in the optimal growth state period and recording as DOtop;
(3) in the process of culturing nitrobacteria, when the concentration of dissolved oxygen in a culture system is increased to 50-70% DOmax, and stays for 5-15 minutes, then a feeding system is started, and after the feeding system is started, the feeding system is stopped when the DO concentration is reduced by 20-40 percentage points; and circulating the steps until the culture is finished.
The upper limit value DOmax of the dissolved oxygen concentration in the step (1) of the invention means that the initial culture solution of the nitrobacteria is added into the aeration reactor, and aeration is carried out by adjusting the aeration amount under the condition of not inoculating microorganisms and starting stirring until the DO concentration is not increased any more, thereby determining the upper limit value DOmax of the DO concentration of the culture system and the corresponding aeration amount Q.
In step (1) of the present invention, the composition of the initial culture solution comprises nitrogen ammonia and trace elements, wherein the substance providing nitrogen ammonia may be at least one of ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium bicarbonate, ammonium carbonate, and the like. The nitrogen concentration is not more than 300mg/L, and the trace elements may include K+、Mg2+、Fe2+、Ca2+、HPO4 2-And H2PO4 -Etc. in a total ion concentration of 0.3 to 10 g/L.
Determining the optimal growth state period of the nitrifying bacteria in the step (2) and adopting a constant aeration quantity Q in the culture process of the bacteria in the step (3).
The dissolved oxygen concentration DOtop in the optimal growth state period in the step (2) of the invention means that the DO concentration is gradually reduced after the nitrobacteria microorganism is inoculated and cultured until the DO is about to be the lowest and stable for 1-2h, the period is the optimal active growth state period of the nitrobacteria, and the DO concentration is controlled at 15% -25% DOmax.
In the present invention, DOmax is determined by adjusting the aeration amount by the aeration system under the condition of starting stirring, and the concentration of DOmax is generally 6-9 mg/L.
In the invention, the aeration reactor is a closed reactor with aeration and stirring functions at the bottom.
In the invention, the nitrobacteria culture medium is determined according to the specific microorganism variety, and the inoculation amount is 0.5-5% of the volume of the initial culture solution.
The nitrifying bacteria inoculated by the method can be any bacteria subjected to enrichment culture known in the field, such as nitrifying bacteria which can be subjected to enrichment culture by adopting CN 201410585640.3.
In the invention, the material supplemented by the feeding system is an inorganic culture solution, and mainly comprises a substance capable of providing ammonia nitrogen, such as an inorganic ammonium compound, wherein the inorganic ammonium compound can be at least one of ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium bicarbonate, ammonium carbonate and the like. The ammonia nitrogen concentration in the inorganic culture solution is 1000-5000 mg/L.
In the invention, the culture conditions of the nitrifying bacteria are as follows: the pH value is 7-9, and the temperature is 25-38 ℃.
Compared with the prior art, the invention has the following advantages:
1. the inventor researches and discovers that under the condition of keeping the ventilation quantity unchanged, the feeding time is controlled by depending on DO concentration within a certain DO concentration range, and the resistance of thalli to dissolved oxygen is domesticated by depending on the concentration mutation of the dissolved oxygen in the culture process, so that the obtained thalli not only has high activity and high propagation speed, but also can well solve the influence of the dissolved oxygen on the activity of the thalli, and still has good nitrification effect when the dissolved oxygen concentration is high under the condition of low load in industry.
2. The method can realize the automatic control culture of the nitrobacteria and is suitable for industrial large-scale application.
Detailed Description
The method and effects of the present invention will be described in further detail by examples. The embodiments are implemented on the premise of the technical scheme of the invention, and detailed implementation modes and specific operation processes are given, but the protection scope of the invention is not limited by the following embodiments.
In the present invention, the upper limit of the dissolved oxygen concentration in the culture system is denoted as DOmax (i.e., the saturated dissolved oxygen concentration in the initial culture solution), and each dissolved oxygen concentration is represented by a relative value to DOmax, and the percentage of increase or decrease is the percentage of the difference between the concentration of a certain DO concentration to DOmax and the concentration of another DO concentration to DOmax. For example, the DO concentration is reduced by 30 percentage points when the feed system is started and controlled to be 60% DOmax and when the feed system is stopped and controlled to be 30% DOmax.
In the invention, the dissolved oxygen is measured by a WTW-300i dissolved oxygen on-line instrument, and the pH value is measured by a Hanna on-line measuring instrument.
The culture method for improving the resistance of nitrobacteria provided by the invention comprises the following specific processes:
(1) firstly, adding a nitrifying bacteria initial culture solution into an aeration reactor, adjusting aeration amount under the conditions of not inoculating microorganisms and starting stirring to perform aeration until the DO concentration is not increased any more, thereby determining the upper limit value DOmax of the DO concentration of a culture system and the corresponding aeration amount Q, and maintaining the aeration amount Q unchanged in the whole bacteria culture process;
(2) inoculating nitrobacteria microorganisms according to 0.5-5% of the volume of the initial culture solution, and gradually reducing the DO concentration after culture until the DO tends to be lowest and stable for 1-2 hours, wherein the DO concentration is the optimal active growth state period of the microorganisms, the DO concentration at the moment is controlled at 15-25% DOmax, and the dissolved oxygen concentration of the nitrobacteria in the optimal growth state period is determined and recorded as DOtop;
(3) in the process of culturing nitrobacteria, when the concentration of dissolved oxygen in a culture system is increased to 50-70% DOmax, and stays for 5-15 minutes, then a feeding system is started, and after the feeding system is started, the feeding system is stopped when the DO concentration is reduced by 20-40 percentage points; and circulating the steps until the culture is finished.
The experimental procedures in the following examples are, unless otherwise specified, conventional in the art. The test materials used in the following examples were purchased from biochemical reagent stores unless otherwise specified.
In the examples and comparative examples of the present invention, the ammonia nitrogen concentration was measured by GB7478-87 "Water quality-ammonium measurement-distillation and titration method"; the ammonia nitrogen removal rate refers to the amount of ammonia nitrogen that can be removed in unit volume of unit time, and the ammonia nitrogen removal rate = (inlet ammonia nitrogen concentration-outlet ammonia nitrogen concentration)/culture time.
EXAMPLE 1 Nitrification bacteria culture
The culture of nitrifying bacteria was carried out in a 100L aeration reactor. The reactor is simultaneously provided with an automatic control system for stirring, pH and feeding. The temperature during the cultivation was set at 32 ℃ and the pH was set at 7.6-7.7.
(1) 50L of microorganism culture medium is firstly added into the aeration reactor, the aeration system is opened and the aeration amount is adjusted to carry out aeration without inoculating microorganisms until the DO concentration does not increase any more, so that the upper limit value DOmax of the DO concentration required by culture and the corresponding aeration amount are determined, and the aeration amount is maintained to be constant in the whole culture process. Wherein the microbial culture medium comprises the following components in percentage by weight: 50g of ammonium sulfate, 1.5g of ferrous sulfate, 0.5g of calcium chloride, 7g of magnesium sulfate heptahydrate and 7g of potassium dihydrogen phosphate.
(2) Inoculating the nitrifying bacteria obtained by the enrichment method of the No. 3 reactor in CN201410585640.3 example 2 according to 1% of the initial culture system, wherein the dissolved oxygen in the system begins to decrease, and finally the DO tends to be lowest and is stabilized at 25% DOmax, and the nitrifying bacteria enter the optimal active growth state period;
(3) in the nitrobacteria culture process, when DO rises to 70% DOmax and stays for 5 minutes, starting the feeding system, adding culture solution with ammonia nitrogen concentration of 1000mg/L, beginning to show a descending trend along with the dissolved oxygen concentration in the substrate adding system, and stopping feeding when DO drops by 40 percentage points; and circulating the steps until the culture system reaches 80% of the total volume of the reactor, turning off the automatic material supplementing system, ending the culture of the batch when the ammonia nitrogen concentration in the culture solution is exhausted, stopping aeration and stirring, discharging supernatant after sedimentation, and harvesting the thalli.
The ammonia oxidation rate of the harvested bacteria is improved to 67 mg/(L.h) from 10 mg/(L.h) during inoculation, and the tolerable dissolved oxygen concentration can reach 8 mg/L.
EXAMPLE 2 Nitrification of bacteria culture
The culture of nitrifying bacteria was carried out in a 100L aeration reactor. The reactor is simultaneously provided with an automatic control system for stirring, pH and feeding. The temperature during the culture was set at 30 ℃ and the pH was set at 7.8 to 7.9.
(1) 50L of microorganism culture medium is firstly added into the aeration reactor, the aeration system is opened and the aeration amount is adjusted to carry out aeration without inoculating microorganisms until the DO concentration does not increase any more, so that the upper limit value DOmax of the DO concentration required by culture and the corresponding aeration amount are determined, and the aeration amount is maintained to be constant in the whole culture process. Wherein the microbial culture medium comprises the following components in percentage by weight: 50g of ammonium sulfate, 1.5g of ferrous sulfate, 0.5g of calcium chloride, 7g of magnesium sulfate heptahydrate and 7g of potassium dihydrogen phosphate.
(2) Inoculating the nitrifying bacteria obtained by the enrichment method of the No. 3 reactor in CN201410585640.3 example 2 according to 3% of an initial culture system, wherein the dissolved oxygen in the system begins to decrease, and finally the DO tends to be lowest and is stabilized at 20% DOmax, and the nitrifying bacteria enter an optimal active growth state period;
(3) in the process of culturing nitrobacteria, when DO rises to 60% DOmax and stays for 10 minutes, starting a feeding system, and adding culture solution with the ammonia nitrogen concentration of 2500 mg/L; the dissolved oxygen concentration in the system begins to show a descending trend along with the substrate supplement, and the feed is stopped when the DO is reduced by 35 percentage points; and circulating the steps until the culture system reaches 80% of the total volume of the reactor, turning off the automatic material supplementing system, ending the culture of the batch when the ammonia nitrogen concentration in the culture solution is exhausted, stopping aeration and stirring, discharging supernatant after sedimentation, and harvesting the thalli.
The ammonia oxidation rate of the harvested bacteria is improved from 11 mg/(L.h) during inoculation to 68 mg/(L.h), and the tolerable dissolved oxygen concentration can reach 7 mg/L.
EXAMPLE 3 Nitrification bacteria culture
The culture of nitrifying bacteria was carried out in a 100L aeration reactor. The reactor is simultaneously provided with an automatic control system for stirring, pH and feeding. The temperature during the culture was set at 35 ℃ and the pH was set at 7.9-8.1.
(1) 50L of microorganism culture medium is firstly added into the aeration reactor, the aeration system is opened and the aeration amount is adjusted to carry out aeration without inoculating microorganisms until the DO concentration does not increase any more, so that the upper limit value DOmax of the DO concentration required by culture and the corresponding aeration amount are determined, and the aeration amount is maintained to be constant in the whole culture process. Wherein the microbial culture medium comprises the following components in percentage by weight: 50g of ammonium sulfate, 1.5g of ferrous sulfate, 0.5g of calcium chloride, 7g of magnesium sulfate heptahydrate and 7g of potassium dihydrogen phosphate.
(2) Inoculating the nitrifying bacteria obtained by the enrichment method of the No. 3 reactor in CN201410585640.3 example 2 according to 5% of an initial culture system, wherein the dissolved oxygen in the system begins to decrease, and finally the DO tends to be lowest and is stabilized at 15% DOmax, and the nitrifying bacteria enter an optimal active growth state period;
(3) in the process of culturing nitrobacteria, when DO rises to 50% DOmax and stays for 15 minutes, starting a feeding system, and adding culture solution with the ammonia nitrogen concentration of 5000 mg/L; the dissolved oxygen concentration in the system begins to show a descending trend again along with the substrate supplement, and the feed is stopped when the DO is reduced by 25 percentage points; and circulating the steps until the culture system reaches 80% of the total volume of the reactor, turning off the automatic material supplementing system, ending the culture of the batch when the ammonia nitrogen concentration in the culture solution is exhausted, stopping aeration and stirring, discharging supernatant after sedimentation, and harvesting the thalli.
The ammonia oxidation rate of the harvested bacteria is increased from 11 mg/(L.h) during inoculation to 69 mg/(L.h), and the tolerable dissolved oxygen concentration can reach 6 mg/L.
Comparative example 1
The difference from example 1 is that: the dissolved oxygen concentration is controlled to be 1-3mg/L in the whole culture process, and the equal amount of the substrate adopts a fed-batch mode. The ammonia nitrogen removal rate of the harvested bacteria is only 40 mg/(L.h).
Comparative example 2
The difference from example 1 is that: step (3) of the culture Process in the nitrobacteria culture process, when DO is increased to 70% DOmax, the feeding system is directly started without staying for a certain time, and the ammonia oxidation rate of the harvested bacterial cells is only 45 mg/(L.h) under the condition that the dissolved oxygen concentration is 8 mg/L.
Example 4 application of nitrifying bacteria
Five reactors which are respectively filled with 300mL of aqueous solution are named as No. I, No. II, No. III, No. IV and No. V, nitrobacteria obtained according to the enrichment method of the No. 3 reactor in CN201410585640.3 example 2 and 200mL of nitrobacteria in the invention examples 1-3 and comparative example 2 are respectively added with the same concentration, aeration is carried out for 2h under the condition that the dissolved oxygen concentration is 6mg/L, then the nitrified bacteria are transferred into five 5L reactors with the same number, wastewater with the ammonia nitrogen concentration of 500mg/L in a certain enterprise is treated under the same condition, the ammonia nitrogen concentration is analyzed after 5h, the ammonia nitrogen removal rates are respectively calculated to be 40%, 74%, 72%, 70% and 49%, and therefore, the nitrobacteria obtained by the invention can bear the impact of high dissolved oxygen under the low-load condition.
Claims (9)
1. A culture method for increasing resistance to nitrifying bacteria, comprising:
(1) determining the upper limit value of the dissolved oxygen concentration in the culture system as DOmax and the corresponding aeration quantity Q;
(2) determining the dissolved oxygen concentration of the nitrifying bacteria in the optimal growth state period and recording as DOtop;
(3) in the process of culturing nitrobacteria, when the concentration of dissolved oxygen in a culture system is increased to 50-70% DOmax, and stays for 5-15 minutes, then a feeding system is started, and after the feeding system is started, the feeding system is stopped when the DO concentration is reduced by 20-40 percentage points; and circulating the steps until the culture is finished.
2. The method as claimed in claim 1, wherein the upper limit value DOmax of the dissolved oxygen concentration in the step (1) is determined by adding the initial culture solution of nitrifying bacteria into the aeration reactor, and adjusting the aeration amount to perform aeration without inoculating microorganisms and turning on stirring until the DO concentration does not increase any more, thereby determining the upper limit value DOmax of the DO concentration of the culture system and the corresponding aeration amount Q.
3. The method according to claim 1, wherein the concentration of ammonia nitrogen in the initial broth is not more than 300 mg/L.
4. The method according to claim 1, wherein a constant aeration rate Q is used during the determination of the optimal growth state period of the nitrifying bacteria in step (2) and the cultivation of the bacteria in step (3).
5. The method as claimed in claim 1, wherein the dissolved oxygen concentration DOtop in the optimal growth state period in the step (2) is that the DO concentration is gradually reduced after the nitrifying bacteria microorganism is inoculated and cultured until the DO is about to be lowest and stable for 1-2h, and the DO concentration is controlled at 15% -25% DOmax in the optimal active growth state period of the nitrifying bacteria.
6. The process according to claim 1, wherein said DOmax is determined by adjusting the aeration rate by the aeration system with agitation turned on, and has a concentration of 6-9 mg/L.
7. The method of claim 1, wherein the nitrifying bacteria culture medium is determined according to the specific microorganism species used, and the inoculation amount is 0.5-5% of the volume of the initial culture solution.
8. The method as claimed in claim 1, wherein the culture medium supplemented by the feeding system is an inorganic culture medium mainly comprising ammonia nitrogen as a substance capable of providing ammonia nitrogen, and the concentration of ammonia nitrogen in the inorganic culture medium is 1000-5000 mg/L.
9. The method according to claim 1, wherein the nitrifying bacteria are cultured under conditions of: the pH value is 7-9, and the temperature is 25-38 ℃.
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