AU2021102864A4 - Ammonia oxidizing bacteria culture medium and preparation method thereof - Google Patents
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- 241001453382 Nitrosomonadales Species 0.000 title claims abstract description 74
- 239000001963 growth medium Substances 0.000 title claims abstract description 64
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims abstract description 56
- 235000017557 sodium bicarbonate Nutrition 0.000 claims abstract description 28
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims abstract description 28
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 24
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims abstract description 18
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 14
- 235000019796 monopotassium phosphate Nutrition 0.000 claims abstract description 14
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims abstract description 14
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 9
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 9
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 9
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 9
- 239000001110 calcium chloride Substances 0.000 claims abstract description 9
- 229910001628 calcium chloride Inorganic materials 0.000 claims abstract description 9
- 235000011148 calcium chloride Nutrition 0.000 claims abstract description 9
- 229910000365 copper sulfate Inorganic materials 0.000 claims abstract description 9
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims abstract description 9
- 239000011790 ferrous sulphate Substances 0.000 claims abstract description 9
- 235000003891 ferrous sulphate Nutrition 0.000 claims abstract description 9
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims abstract description 9
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims abstract description 9
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims abstract description 9
- 235000019341 magnesium sulphate Nutrition 0.000 claims abstract description 9
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims abstract description 9
- 235000019799 monosodium phosphate Nutrition 0.000 claims abstract description 9
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims abstract description 9
- 235000011121 sodium hydroxide Nutrition 0.000 claims abstract description 9
- 239000000203 mixture Substances 0.000 claims description 23
- 239000000243 solution Substances 0.000 claims description 18
- 230000001954 sterilising effect Effects 0.000 claims description 18
- 238000004659 sterilization and disinfection Methods 0.000 claims description 18
- -1 ION sodium hydroxide Chemical class 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 3
- 239000012498 ultrapure water Substances 0.000 claims description 3
- 239000004695 Polyether sulfone Substances 0.000 claims description 2
- 229920006393 polyether sulfone Polymers 0.000 claims description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 abstract description 29
- 230000012010 growth Effects 0.000 abstract description 26
- 229910000029 sodium carbonate Inorganic materials 0.000 abstract description 14
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 11
- 229910052799 carbon Inorganic materials 0.000 abstract description 11
- 235000015097 nutrients Nutrition 0.000 abstract description 7
- 230000004060 metabolic process Effects 0.000 abstract description 4
- 238000010521 absorption reaction Methods 0.000 abstract description 3
- 230000003139 buffering effect Effects 0.000 abstract description 3
- 230000000052 comparative effect Effects 0.000 description 28
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 16
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 14
- 241000894006 Bacteria Species 0.000 description 12
- 241000605121 Nitrosomonas europaea Species 0.000 description 12
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 8
- 229910021529 ammonia Inorganic materials 0.000 description 7
- 238000009472 formulation Methods 0.000 description 7
- 229910052757 nitrogen Inorganic materials 0.000 description 7
- 230000003647 oxidation Effects 0.000 description 7
- 238000007254 oxidation reaction Methods 0.000 description 7
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 6
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000004520 electroporation Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 229910002651 NO3 Inorganic materials 0.000 description 3
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 229910000019 calcium carbonate Inorganic materials 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 2
- IOVCWXUNBOPUCH-UHFFFAOYSA-N Nitrous acid Chemical compound ON=O IOVCWXUNBOPUCH-UHFFFAOYSA-N 0.000 description 2
- JVMRPSJZNHXORP-UHFFFAOYSA-N ON=O.ON=O.ON=O.N Chemical compound ON=O.ON=O.ON=O.N JVMRPSJZNHXORP-UHFFFAOYSA-N 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 229910001873 dinitrogen Inorganic materials 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000010865 sewage Substances 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 108010061397 Ammonia monooxygenase Proteins 0.000 description 1
- 108010086710 Hydroxylamine dehydrogenase Proteins 0.000 description 1
- 241000605122 Nitrosomonas Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000012365 batch cultivation Methods 0.000 description 1
- 235000019577 caloric intake Nutrition 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000012851 eutrophication Methods 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
Classifications
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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
- C12N1/20—Bacteria; Culture media therefor
-
- 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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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to ammonia oxidizing bacteria culture medium and preparation
method thereof. One liter of the ammonia oxidizing bacteria culture medium includes following
components by mass concentration:3.3 g/L of ammonium sulfate, 5.88 g/L of potassium
dihydrogen phosphate, 0.0879 g/L of magnesium sulfate, 0.0201 g/L of calcium chloride,
0.00152 g/L of ferrous sulfate, 0.851x10-4 g/L of copper sulfate, 0.4667 g/L of sodium
dihydrogen phosphate, 2.67 g/L of sodium hydroxide and 1.8-2.2 g/L of sodium bicarbonate.
Compared with traditional ammonia oxidizing bacteria culture medium, the types of inorganic
carbon sources are improved. Replacing sodium carbonate with sodium bicarbonate can provide
ammonia oxidizing bacteria with a richer carbon source and a stronger buffering ability. The
ammonia oxidizing bacteria culture medium provided by the present invention can promote the
absorption of nutrients by ammonia oxidizing bacteria and facilitate relevant metabolic
processes, which benefits the stable growth and reproduction of the ammonia oxidizing
bacteria.
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-LI-Example 1
0.05 -- M-Comparative example 1
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Figure 1
Description
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0.06 -LI-Example 1
0.05 -- M-Comparative example 1
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Figure 1
Ammonia Oxidizing Bacteria Culture Medium and Preparation Method Thereof
Technical Field
[0001] The present invention relates to the technical field of microorganisms, and particularly relates to an ammonia oxidizing bacteria culture medium and a preparation method thereof.
Background
[0002] Ammonia nitrogen is one of the most important pollutants in water bodies, which can lead to water eutrophication. With the development of industry and agriculturee, the pollution of ammonia nitrogen has become more and more serious, which has become one of the urgent problems to be solved in the sewage treatment area and also one of the key environmental indicators. Ammonia oxidizing bacteria remove ammonia nitrogen through ammonia oxidation. The ammonia nitrogen is first converted to hydroxylamine by ammonia monooxygenase, and then hydroxylamine is oxidized to nitrite by hydroxylamine oxidase. Next, nitrite is converted into nitrate by nitrite oxidizing bacteria, and then nitrate is gradually reduced to nitrogen gas by denitrifying bacteria and finally nitrogen gas is released from the water, thereby complete denitrification is achieved. Moreover, nitrate/ nitrite can also be directly removed by partial denitrification or anaerobic ammonia oxidation. Therefore, the metabolism of ammonia oxidizing bacteria is a key step in biological denitrification.
[0003] Researchers discovered the phenomenon of ammonia oxidation in 1895. Since the middle of the last century, studies on the physiological and biochemical characteristics of ammonia oxidizing bacteria and their application have never stopped. However, there is no large-scale industrial application of ammonia oxidizing bacteria. The reason is that ammonia oxidizing bacteria are chemoautotrophic bacteria that grow slowly and are sensitive to factors such as temperature, pH, dissolved oxygen and heavy metals. Ammonia oxidizing bacteria are extremely difficult to be cultivated and purified. In the sewage treatment system, such kind of bacteria is difficult to form the dominant flora and is easily affected by environmental factors, which weakens the ability of nitrogen removal in system. The products of the ammonia oxidation process by ammonia oxidizing bacteria are nitrite and hydrogen ions. The accumulation of hydrogen ions in the culture medium will cause the decrease of pH. And the hydrogen ions combine with nitrite to form free nitrous acid. During batch cultivation, the growth of ammonia oxidizing bacteria in the culture medium will be affected by low pH and high concentration of free nitrous acid and will be stopped finally. Therefore, in the cultivation process of ammonia oxidizing bacteria, the requirements for the culture medium are extremely strict. The nutrient ingredients of the culture medium has an important influence on the growth, reproduction of the bacteria and the transformation rate of ammonia nitrogen. It not only promote ammonia oxidizing bacteria to grow rapidly after transformation, but also help ammonia oxidizing bacteria to make more use of the nutrients in the culture medium to obtained a large number of bacteria eventually. A proper formulation of culture medium will have a great impact on the growth of ammonia oxidizing bacteria.
[0004] Currently, the composition of the culture medium of ammonia oxidizing bacteria is basically based on the culture medium formulation of Nitrosomonas europaea provided by the American Culture Collection (https://www.atcc.org/Products/All/19718.aspx, ATCC medium: 2265). The formulation uses sodium carbonate at a concentration of 0.4 g/L as an inorganic carbon source, and the initial alkalinity of sodium carbonate in the culture medium is 0.378 g/L (calculated as calcium carbonate). Since the first step ionization of carbonic acid is much stronger than the second step ionization, the capacity of hydrolysis of sodium carbonate and the ability to increase pH are greater than that of sodium bicarbonate which can also be used as an inorganic carbon source. The sodium carbonate in the formulation also increases the alkalinity and pH of the culture medium as well as providing the carbon source for the culture medium. In order to keep the initial pH of the culture medium within a suitable range for the growth of ammonia oxidizing bacteria, the dosage of sodium carbonate should not be too large. When lg of ammonia nitrogen is oxidized by ammonia oxidizing bacteria, 7.14g of alkalinity will be consumed to balance the H+ produced by the ammonia oxidation process. Since the amount of sodium carbonate addition in the medium is limited by the initial pH which should not be too high, the alkalinity in the medium is limited. As the alkalinity is consumed during the growth of ammonia oxidizing bacteria, the pH will quickly drop to a range that inhibits the growth of ammonia oxidizing bacteria. Consequently, the nutrient utilization rate of ammonia oxidizing bacteria would be low and the growth of ammonia oxidizing bacteria would be slow.
[0005] Therefore, from the perspective of the types of inorganic carbon sources in the culture medium, it has a great value in the cultivation and application of ammonia oxidizing bacteria to develop a culture medium that can make ammonia oxidizing bacteria grow stably and rapidly so as to utilize the nutrients to a greater extent.
Summary
[0006] Based on the problems in the prior art and after extensive studies, the present inventors found that the cultivation of ammonia oxidizing bacteria can be stable and rapid. Also, the longer growth period which is unexpected can be achieved under the cultivation of the culture medium invented in this invention. The present invention is completed thereby.
[0007] Specifically, the present invention includes the following technical solutions.
[0008] In one aspect, a kind of ammonia oxidizing bacteria culture medium is provided in the present invention, and the components of the ammonia oxidizing bacteria culture medium in each liter include (mass concentration): ammonium sulfate 3.3g/L, potassium dihydrogen phosphate 5.88 g/L, magnesium sulfate 0.0879 g/L, calcium chloride 0.0201 g/L, ferrous sulfate 0.00152 g/L, copper sulfate 0.851x10-4 g/L, sodium dihydrogen phosphate 0.4667 g/L, sodium hydroxide 2.67 g/L, and sodium bicarbonate 1.8-2.2 g/L.
[0009] The ammonia oxidizing bacteria culture medium of the present invention can provide alkalinity as well as carbon source by using sodium bicarbonate. Compared with sodium carbonate, sodium bicarbonate has a lower alkalinity. Considering the initial pH of the culture medium which should be suitable for the growth of ammonia oxidizing bacteria, the dosage of sodium bicarbonate can be higher than that of sodium carbonate. Sodium bicarbonate can not only combine with the OH- previously in the medium but also with the H' produced by ammonia oxidation of ammonia oxidizing bacteria. When sodium bicarbonate is selected as the carbon source, it can provide a richer carbon source and a stronger buffering capacity, which is beneficial to the growth of ammonia oxidizing bacteria and the utilization of nitrogen sources in the culture medium. Furthermore, the addition of ammonium sulfate can provide rich nitrogen source for the growth of ammonia oxidizing bacteria. The addition of potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, ferrous sulfate and copper sulfate can promote the absorption of nutrients by ammonia oxidizing bacteria and the related metabolic processes. By adding potassium dihydrogen phosphate, sodium dihydrogen phosphate and sodium hydroxide, a buffer system can be formed together with sodium bicarbonate, so as to maintain the acid-base balance of the culture medium. By using the ammonia oxidizing bacteria culture medium of the present invention, an excellent ammonia oxidizing bacteria culture medium with wide applicability can be obtained in the present invention.
[0010] The ammonia oxidizing bacteria culture medium of the present invention has a good effect especially on cultivating Nitrosomonas europaea (a typical model strain of ammonia oxidizing bacteria), and it can improve the growth of the bacteria to obtain a higher final concentration of bacterial with a high degree of utilization of the nitrogen source in the culture medium.
[0011] In another aspect, the present invention provides a preparation method of the above ammonia oxidizing bacteria culture medium, including the following steps:
(1) For each liter of water, add 4.125 g of ammonium sulfate, 0.5167 g of potassium dihydrogen phosphate, 0.0251 g of magnesium sulfate and 0.0019 g of calcium chloride, and then add 2.28 mg of ferrous sulfate and 1.07 mg of copper sulfate followed by mixing and stirring;
(2) Mix potassium dihydrogen phosphate, sodium dihydrogen phosphate and ultrapure water in the ratio of 8.2:0.7:300 by weight, and stir evenly;
(3) Add ION sodium hydroxide to the mixed solution obtained in step (2) until the pH of the solution reaches 7.8-8.2;
(4) Mix the solution obtained from the step (1) with the solution obtained from the step (3) in a ratio of 4:1 by volume followed by sterilization
(5) Sterilize the sodium bicarbonate solution having a concentration of 66.67 g/L, and add it to the mixture obtained in step (4) to make the final concentration of sodium bicarbonate 1.8-2.2 g/L, mix evenly to obtain the ammonia oxidizing bacteria culture medium.
[0012] According to the preparation method of the ammonia oxidizing bacteria culture medium of the present invention, preferably, the sterilization in the step (4) is high temperature and high pressure sterilization, and the sterilization time is 15-30 minutes, the sterilization temperature is 121-122°C.
[0013] According to the preparation method of the ammonia oxidizing bacteria culture medium of the present invention, preferably, the sterilization in the step (5) is filter membrane sterilization by filtration, and the filter membrane used in the sterilization is a 0.22 m or 0.45 m polyethersulfone filter membrane.
[0014] Compared with the prior art, the advantages of the present invention areas follows:
[0015] In the ammonia oxidizing bacteria culture medium provided by the present invention, sodium bicarbonate is used as a richer carbon source, which can provide a richer carbon source as well as an stronger buffering capacity, and is beneficial to the growth of ammonia oxidizing bacteria and the utilization of nitrogen source in the culture medium. The use of potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, ferrous sulfate and copper sulfate can promote the absorption of nutrients and the related metabolic processes of ammonia oxidizing bacteria, which is advantageous to stimulate the stable growth and reproduction of ammonia oxidizing bacteria. Meanwhile, ammonium sulfate provides a rich source of nitrogen for the growth of ammonia oxidizing bacteria and ensures sufficient energy intake for ammonia oxidizing bacteria. Potassium dihydrogen phosphate, sodium dihydrogen phosphate, sodium hydroxide and sodium bicarbonate together form a buffer system which provides a stable pH for the growth of ammonia oxidizing bacteria. Therefore, the ammonia oxidizing bacteria culture medium of the present invention can promote a good growth of ammonia oxidizing bacteria and display a good application prospect.
[0016] According to the preparation method of the ammonia oxidizing bacteria culture medium of the present invention, all the rest components of the culture medium can be sterilized under high temperature and high pressure, except for the sodium bicarbonate, which can be decomposed into sodium carbonate under high temperature and high pressure. Compared with culture medium formulation provided by the American Culture Collection for Nitrosomonas europaea, which requires filtration sterilization as a whole, the preparation method of the ammonia oxidizing bacteria culture medium of the present invention is easier to operate, and the ammonia oxidizing bacteria are not easily contaminated.
Brief Description of Drawings
[0017] Figure 1 shows the change of OD 600nm in Example 1 and Comparative Example 1.
[0018] Figure 2 shows the change in the concentration of nitrite nitrogen in Example 1 and Comparative Example 1.
[0019] Figure 3 shows the pH change in Example 1 and Comparative Example 1.
Description of Embodiments
[0020] The technical solution of the present invention will be further described in combination with the drawings and specific embodiments as follow. The skilled in the art should understand that the described embodiments only help understanding the present invention, and should not be regarded as specific limitations to the present invention.
Example 1:
[0021] The preparation of ammonia oxidizing bacteria culture medium including the following components by mass concentration: sodium bicarbonate 2 g/L, ammonium sulfate 3.3 g/L, potassium dihydrogen phosphate 5.88 g/L, magnesium sulfate 0.0879 g/L, calcium chloride 0.0201 g/L, ferrous sulfate 0.00152 g/L, copper sulfate 0.0851 mg/L, sodium dihydrogen phosphate 0.4667 g/L and sodium hydroxide 2.67 g/L.
[0022] Preparation method:
(1) Add 4.125 g of ammonium sulfate, 0.5167 g of potassium dihydrogen phosphate, 0.0251 g of magnesium sulfate and 0.0019 g of calcium chloride to 1 liter of water, and then add 2.28 mg of ferrous sulfate and 1.07 mg of copper sulfate to the above solution followed by mixing and stirring;
(2) Mix potassium dihydrogen phosphate, sodium dihydrogen phosphate and ultrapure water in the ratio of 8.2:0.7:300 by weight, and stir evenly;
(3) Add ION sodium hydroxide to the mixed solution in step (2) until the pH of the solution reaches 8;
(4) Mix the solution in steps (1) and (3) in a ratio of 4:1 by volume evenly followed by sterilization;
(5) Sterilize the sodium bicarbonate solution having a concentration of 66.67 g/L, and add it to the mixture obtained from the step (4) to make the final concentration of sodium bicarbonate 2 g/L. After mixing, the ammonia oxidizing bacteria culture medium is obtained.
Comparative example 1:
[0023] The difference between Example 1 and Comparative Example 1 is that this comparative example does not include sodium bicarbonate, however, 0.6 g of anhydrous sodium carbonate is added to the mixture obtained from the step (4), and the rest are the same as Example 1. After mixing, the ammonia oxidizing bacteria culture medium of Comparative Example 1 is obtained.
[0024] An equal amount of Nitrosomonas europaea (ATCC@ 19718TM) is transferred to Example 1 and Comparative Example 1 for cultivation,respectively. The bacteria are placed in a shaker at 30°C and 180 r/min for shading cultivation. Samplings are taken every 12 hours to record the growth of the bacteria.The OD 600nm value of the bacterial solution, the concentration of nitrite nitrogen as well as the pH of the culture medium are measured. The detailed results are shown in Figures 1 to 3 respectively.
[0025] The alkalinity provided by sodium bicarbonate in Example 1 is 2.383 g/L (calculated as calcium carbonate), while the alkalinity provided by sodium carbonate in Comparative Example 1 is 0.378 g/L (calculated as calcium carbonate). The former is about 6.3 times of the latter. By comparing Example 1 with Comparative Example 1, it can be seen that after replacing sodium carbonate with sodium bicarbonate in the culture medium of the present invention, the initial pH is almost same, but the growth of Nitrosomonas europaea is significantly different. It illustrates that the culture medium provided by the present invention can promote the growth of Nitrosomonas europaea and results in obvious ammonia oxidation effect compared with the traditional ammonia oxidizing bacteria culture medium where sodium carbonate is added. In addition, in the culture medium provided by the present invention, the overall growth period of the bacterial is longer, the utilization rate of nitrogen source is higher, and the amount of bacteria finally obtained is larger. The ammonia oxidizing bacteria culture medium provided by the present invention shows an excellent effect on the cultivation of the ammonia oxidizing bacteria model strain Nitrosomonas europaea.
Example 2:
[0026] The culture medium formulation and the preparation method in Example 2 are the same as those in Example 1.
[0027] Take out 0.1 mL of Nitrosomonas europaea concentrated from Example 1 with the OD value of 2, and transfer it to 500 mL of fresh culture medium of Example 1 after electroporation at 1.2 KV/mm. After 48 hrs of incubation at 30°C and 180 r/min, the ammonia nitrogen consumption in Example 1 is 350 mg/L, and the OD 600nm value is 0.05.
Comparative Example 2:
[0028] The culture medium formulation and the preparation method in Comparative Example 2 are the same as those in Comparative Example 1.
[0029] Take out 0.1 mL of Nitrosomonas europaeaconcentrated from the logarithmic growth phase in Comparative Example 1with the OD value of 2, and transfer it to 500 mL of fresh culture medium of Comparative Example 1 after electroporation at 1.2 KV/mm. After 48 hrs of incubation at 30°C and 180 r/min, the ammonia nitrogen consumption in Comparative Example 1 is 150 mg/L, and the OD 600nm value is about 0.02.
[0030] Compared Example 2 with Comparative Example 2, it can be seen that when the sodium carbonate in the culture medium is replaced with sodium bicarbonate, the resuscitation effect of Nitrosomonas europaea after electroporation is more prominent. The difficulty of the cloning technology for ammonia oxidizing bacteria is its slow growth. Using the culture medium in Example 1 and Example 2 can attain a rapid resuscitation of ammonia oxidizing bacteria after electroporation, which is beneficial to the subsequent processes of the cloning of ammonia oxidizing bacteria.
Comparative Example 3:
[0031] The difference between this comparative example and Example 1 is that in step (5), the sodium bicarbonate solution with the concentration of 66.67 g/L is sterilized and then added to the mixture obtained from the step (4) to make the final concentration of sodium bicarbonate 4 g/L to get Comparative Example 3. The rest are the same as those in Example 1.
Comparative Example 4:
[0032] The difference between this comparative example and Example 1 is that in step (5), the sodium bicarbonate solution with the concentration of 66.67 g/L is sterilized and then added to the mixture obtained from the step (4) to make the final concentration of sodium bicarbonate 6 g/L to get Comparative Example 4. The rest are the same as those in Example 1.
Comparative Example 5:
[0033] The difference between this comparative example and Example 1 is that in step (5), the sodium bicarbonate solution with the concentration of 66.67 g/L is sterilized and then added to the mixture obtained from the step (4) to make the final concentration of sodium bicarbonate 8 g/L to get Comparative Example 5. The rest are the same as those in Example 1.
[0034] Same amount ofNitrosomonas europaea are respectively transferred to Example 1 and Comparative Examples 3, 4, and 5 for cultivation. The bacteria are placed in a shaker at 30°C and 180r/min for shading cultivation, and the growth of the bacteria is recorded. The results proves that only the bacterial amount of Nitrosomonas europaea in Example 1 increases significantly, whereas there is no growth of Nitrosomonas europaea in Comparative Examples 3, 4, or 5. In summary, the sodium bicarbonate content in the ammonia oxidizing bacteria culture medium provided by the present invention is suitable for the growth of Nitrosomonas europaea.
[0035] The applicant declares that the above examples are used to illustrate the microbial culture medium of the present invention, the preparation method as well as the application thereof, and are not used to limit the present invention, i.e., are not suggesting that the present invention must rely on the above detailed methods to be implemented. The skilled in the art should understand that any improvements for the present invention, any equivalent replacement of each raw material of the present invention and the addition of auxiliary components, as well as the selection of specific methods, etc. will fall within the protection scope and disclosure of the present invention.
[0036] It is obvious for the skilled in the art that various improvements and changes can be taken to the specific embodiments of the present specification without departing from the scope or spirit of the present invention. Other embodiments derived from the description of the present invention will be obvious to the skilled. The specification and examples of this application are only exemplary.
Claims (4)
1. An ammonia oxidizing bacteria culture medium, characterized in that, each liter of the ammonia oxidizing bacteria culture medium includes following components by mass concentration: ammonium sulfate 3.3 g/L, potassium dihydrogen phosphate 5.88 g/L, magnesium sulfate 0.0879 g/L, calcium chloride 0.0201 g/L, ferrous sulfate 0.00152 g/L, copper sulfate 0.851x10-4 g/L, sodium dihydrogen phosphate 0.4667 g/L, sodium hydroxide 2.67 g/L, and sodium bicarbonate 1.8-2.2 g /L.
2. A preparation method of the ammonia oxidizing bacteria culture medium according to claim 1, characterized in that, (1) for each liter of water, add 4.125 g of ammonium sulfate, 0.5167 g of potassium dihydrogen phosphate, 0.0251 g of magnesium sulfate and 0.0019 g of calcium chloride, and then add 2.28 mg of ferrous sulfate and 1.07 mg of copper sulfate followed by mixing and stirring; (2) Mix potassium dihydrogen phosphate, sodium dihydrogen phosphate and ultrapure water in the ratio of 8.2:0.7:300 by weight, and stir evenly; (3) Add ION sodium hydroxide to the mixed solution obtained from the step (2) until the pH of the solution reaches 7.8-8.2; (4) Mix the solution obtained from the step (1) with the solution obtained in step (3) in a ratio of 4:1 by volume followed by sterilization (5) Sterilize the sodium bicarbonate solution with the concentration of 66.67 g/L, and add it to the mixture obtained from the step (4) to make the final concentration of sodium bicarbonate 1.8-2.2 g/L and mix to obtain the ammonia oxidizing bacteria culture medium.
3. The preparation method according to claim 2, wherein the sterilization in the step (4) is high temperature and high pressure sterilization, and the sterilization time is 15-30 minutes, the sterilization temperature is 121-122°C.
4. The preparation method according to claim 2 or 3, wherein the sterilization in the step (5) is filter membrane sterilization by filtration, and the filter membrane used in the sterilization
is a 0.22 pm or 0.45 pm polyethersulfone filter membrane.
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