NL2026574B1 - Preliminary screening method of bacteria having an ammonia nitrogen degradation function - Google Patents
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- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 title claims abstract description 83
- 230000015556 catabolic process Effects 0.000 title claims abstract description 44
- 238000006731 degradation reaction Methods 0.000 title claims abstract description 44
- 241000894006 Bacteria Species 0.000 title claims abstract description 42
- 238000012216 screening Methods 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 title claims abstract description 12
- 239000001963 growth medium Substances 0.000 claims abstract description 12
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 8
- 239000012224 working solution Substances 0.000 claims abstract description 7
- 239000002609 medium Substances 0.000 claims abstract description 4
- 238000002798 spectrophotometry method Methods 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 32
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 24
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 18
- 235000019270 ammonium chloride Nutrition 0.000 claims description 12
- 229910052757 nitrogen Inorganic materials 0.000 claims description 12
- 239000011573 trace mineral Substances 0.000 claims description 7
- 235000013619 trace mineral Nutrition 0.000 claims description 7
- 239000008223 sterile water Substances 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- 238000002835 absorbance Methods 0.000 claims description 3
- 239000011609 ammonium molybdate Substances 0.000 claims description 3
- 235000018660 ammonium molybdate Nutrition 0.000 claims description 3
- APUPEJJSWDHEBO-UHFFFAOYSA-P ammonium molybdate Chemical compound [NH4+].[NH4+].[O-][Mo]([O-])(=O)=O APUPEJJSWDHEBO-UHFFFAOYSA-P 0.000 claims description 3
- 229940010552 ammonium molybdate Drugs 0.000 claims description 3
- 230000001580 bacterial effect Effects 0.000 claims description 3
- GFHNAMRJFCEERV-UHFFFAOYSA-L cobalt chloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Co+2] GFHNAMRJFCEERV-UHFFFAOYSA-L 0.000 claims description 3
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- CNFDGXZLMLFIJV-UHFFFAOYSA-L manganese(II) chloride tetrahydrate Chemical compound O.O.O.O.[Cl-].[Cl-].[Mn+2] CNFDGXZLMLFIJV-UHFFFAOYSA-L 0.000 claims description 3
- LJCNRYVRMXRIQR-OLXYHTOASA-L potassium sodium L-tartrate Chemical compound [Na+].[K+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O LJCNRYVRMXRIQR-OLXYHTOASA-L 0.000 claims description 3
- 229940074439 potassium sodium tartrate Drugs 0.000 claims description 3
- 235000011006 sodium potassium tartrate Nutrition 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 claims description 2
- 229940052299 calcium chloride dihydrate Drugs 0.000 claims description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 2
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 claims description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims 2
- 238000002360 preparation method Methods 0.000 claims 2
- 238000005119 centrifugation Methods 0.000 claims 1
- 238000000354 decomposition reaction Methods 0.000 claims 1
- 238000011081 inoculation Methods 0.000 claims 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims 1
- 235000019341 magnesium sulphate Nutrition 0.000 claims 1
- 230000006870 function Effects 0.000 abstract description 15
- 238000002474 experimental method Methods 0.000 abstract description 11
- 230000000813 microbial effect Effects 0.000 abstract description 8
- 239000011550 stock solution Substances 0.000 abstract description 5
- 230000002349 favourable effect Effects 0.000 abstract description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 20
- 241000193830 Bacillus <bacterium> Species 0.000 description 18
- 241000194107 Bacillus megaterium Species 0.000 description 17
- 241000589291 Acinetobacter Species 0.000 description 13
- 241000588748 Klebsiella Species 0.000 description 9
- 241000588770 Proteus mirabilis Species 0.000 description 9
- 229910021529 ammonia Inorganic materials 0.000 description 9
- 244000063299 Bacillus subtilis Species 0.000 description 7
- 235000014469 Bacillus subtilis Nutrition 0.000 description 7
- 241000588697 Enterobacter cloacae Species 0.000 description 5
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 4
- 241000193755 Bacillus cereus Species 0.000 description 4
- 241001014264 Klebsiella variicola Species 0.000 description 4
- 241000588625 Acinetobacter sp. Species 0.000 description 3
- 241000207199 Citrus Species 0.000 description 3
- 241000282376 Panthera tigris Species 0.000 description 3
- 238000009360 aquaculture Methods 0.000 description 3
- 244000144974 aquaculture Species 0.000 description 3
- 235000020971 citrus fruits Nutrition 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 241000122230 Acinetobacter junii Species 0.000 description 2
- 241001135518 Acinetobacter lwoffii Species 0.000 description 2
- 241000194108 Bacillus licheniformis Species 0.000 description 2
- 239000007836 KH2PO4 Substances 0.000 description 2
- 241000283958 Manis javanica Species 0.000 description 2
- 241000289692 Myrmecophagidae Species 0.000 description 2
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 2
- 208000025174 PANDAS Diseases 0.000 description 2
- 208000021155 Paediatric autoimmune neuropsychiatric disorders associated with streptococcal infection Diseases 0.000 description 2
- 240000004718 Panda Species 0.000 description 2
- 235000016496 Panda oleosa Nutrition 0.000 description 2
- 241001076189 Proteus hauseri Species 0.000 description 2
- 241000588767 Proteus vulgaris Species 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000000149 chemical water pollutant Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 229940007042 proteus vulgaris Drugs 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- TVZRAEYQIKYCPH-UHFFFAOYSA-N 3-(trimethylsilyl)propane-1-sulfonic acid Chemical compound C[Si](C)(C)CCCS(O)(=O)=O TVZRAEYQIKYCPH-UHFFFAOYSA-N 0.000 description 1
- 241000881711 Acipenser sturio Species 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 235000006576 Althaea officinalis Nutrition 0.000 description 1
- 241000749858 Ara macao Species 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 241001150381 Bacillus altitudinis Species 0.000 description 1
- 241000278457 Bacillus siamensis Species 0.000 description 1
- 241000012248 Bacillus toyonensis Species 0.000 description 1
- 241000555281 Brevibacillus Species 0.000 description 1
- 241001253191 Cacatua galerita Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000282461 Canis lupus Species 0.000 description 1
- 241000879755 Caracal Species 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- 241000749894 Eclectus roratus Species 0.000 description 1
- 241000282555 Erythrocebus patas Species 0.000 description 1
- 241000282575 Gorilla Species 0.000 description 1
- 241000282821 Hippopotamus Species 0.000 description 1
- 241000588747 Klebsiella pneumoniae Species 0.000 description 1
- 241000830586 Machlolophus spilonotus Species 0.000 description 1
- 241000289572 Macropus Species 0.000 description 1
- 241000289581 Macropus sp. Species 0.000 description 1
- 101100328463 Mus musculus Cmya5 gene Proteins 0.000 description 1
- 241000282317 Paguma larvata Species 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 241000615105 Psittacula alexandri Species 0.000 description 1
- 241000577388 Psittacula derbiana Species 0.000 description 1
- 241000578498 Psittacus erithacus Species 0.000 description 1
- 241001232824 Pyrrhura molinae Species 0.000 description 1
- 241000282806 Rhinoceros Species 0.000 description 1
- 241000427251 Sabaeus Species 0.000 description 1
- 241000282696 Saimiri sciureus Species 0.000 description 1
- 241001504624 Streptopelia Species 0.000 description 1
- 241000287181 Sturnus vulgaris Species 0.000 description 1
- 241000565707 Tapirus bairdii Species 0.000 description 1
- 241000283907 Tragelaphus oryx Species 0.000 description 1
- 241000289674 Vombatidae Species 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 229960005069 calcium Drugs 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 239000002781 deodorant agent Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- NSNHWTBQMQIDCF-UHFFFAOYSA-N dihydrate;hydrochloride Chemical compound O.O.Cl NSNHWTBQMQIDCF-UHFFFAOYSA-N 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- RVUXIPACAZKWHU-UHFFFAOYSA-N sulfuric acid;heptahydrate Chemical compound O.O.O.O.O.O.O.OS(O)(=O)=O RVUXIPACAZKWHU-UHFFFAOYSA-N 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a preliminary screening method of bacteria having an ammonia nitrogen degradation function, comprising preparing an ammonia nitrogen selective culture medium, preparing an ammonia nitrogen standard stock solution, preparing an ammonia nitrogen standard working solution, measuring absor’oance, inoculating strains, detecting the 10 ammonia nitrogen content of the ammonia nitrogen selective cuiture medium by using a _ Nessler's reagent spectrophotometry method, wherein the ammonia nitrogen degradation rate : . (the ammonia nitrogen content in a control group …… the ammonia nitrogen content in an experimental group) / the ammonia nitrogen content in the control group. According to the method disclosed in the invention, the bacteria {with different ammonia nitrogen degradation . 15 functions in microbial bacteria can be screened out, this is notavailable in the existing experimental methods, so that a favorable guarantee is provided for a subsequent bacteria differentiate experiment, the gap of existing experimental screening of bacteria having an Ê ammonia nitrogen degradation function is filled, and the ammonia nitrogen degradation experimental method is further improved.
Description
PRELIMINARY SCREENING METHOD OF BACTERIA HAVING AN AMMONIA | NITROGEN DEGRADATION FUNCTION | FIELD OF THE INVENTION |
[0001] The invention relates to the technical field of microbial bacterial screening, in particular to a preliminary screening method of bacteria having an ammonia nitrogen | degradation function. | BACKGROUND OF THE INVENTION î |
[0002] In the field of microbial bacteria, Bacillus is widely distributed and has strong adaptability. Wherein Bacillus subtilis and Bacillus licheniformis are cornmon probiotics | having multiple biological functions. The prior arts closest to the present invention | include:
[0003] (1) Liu Shubin, Wang Xinrui, Lin Zhuangqi, Cai Yan, Wu Yue, Liu Xiang, Zhou Yongcan, Wang Shifeng. Study on water purification ability of Bacillus subtilis HAINUP40 [J]. Fisheries science, 2018, 37 (02): 159-166. The experiment using an | ammonia nitrogen degradation screening culture medium showed that Bacillus subtilis HAINUP40 had a significant effect on ammonia nitrogen degradation, and the ammonia nitrogen removal rate reached the highest value of 57.58% on the fourth day of the | experiment. |
[0604] (2) Chen Jinhao, Zheng Jinbin, Shi Tianyi, Li Tianjiao, Wang Panpan, Mao Yong, Su | Yongquan, Wang Jun, Isolation and identification of a Bacillus strain degrading ammonia nitrogen from prawn cultural water and optimization of fermentation formulation thereof | IJ]. Journal of Fisheries Research, 2019, 41{03): 175-186. The isolated Bacillus subtilis DSS degraded ammonia nitrogen rapidly in 5 days, and reached the maximum degradation rate of (36.41 + 0.07)%, and the ammonia nitrogen remained stable at about 36% in the last 3 days. |
[0005] (3) Hou Ying, Sun Junde, Xu Jiangiang, Xu Chaole:. Study on the ammonia nitrogen degradation characteristics of Bacillus megaterium in aquaculture water [J]. Journal of Shenyang Agricultural University, 2006(04): 607-610. Study on the ammonia nitrogen degradation characteristics of Bacillus megaterium. When the initial concentration of the ammonia nitrogen is below 50 mg’ L*, the ammonia nitrogen degradation rate of strain X2 | can reach more than 95% within 24 h.
[0006] (4) Kuang Qun, Sun Mei, Zhang Weina, Chen Qiuhong, Gao Liang, Shi Dalin, Hu | Ling. The biological characteristics of Bacillus megaterium JSSW-JD and its effect on | nitrogen and phosphorus in aquaculture water [J]. Jiangsu Agricultural Sciences, 2013, | 41(04): 222-225. It is concluded that Bacillus megaterium JSSW-JD has a strong ability to | degrade nitrite in aquaculture water. After 8 days of treatment of fish pond water, reservoir | water, and crab pond water, the degradation rate of nitrite reached 98.1%, 94.9%, 67.8%. | Bacillus megaterium JSSW-JD does not have a significant degradation effect on ammonia nitrogen, |
[0007] (5) Zhang Chao, Shang Rundong, Liu Yuxuan, Yang Guoging, Li Lu, Shi Lei, Jin Yongsheng. Screening of strain SDB 1-2 and its application in treatment of landfill leachate | [J]. Journal of Beijing University OF Agriculture, 2019, 34(02): 1-4. Klebsiella pneumoniae SDB 1-2 isolated from the landfill leachate was found that it can degrade man- made wastewater with an initial ammonia nitrogen concentration of up to 2000 mg/L. The | highest removal rates of ammonia nitrogen, TN and COD were 51.15, %, 35.03%, 57 A45%|[5]. à |
[0008] In the above mentioned prior arts, although the ammonia nitrogen degradation function | of microbial bacteria can be achieved, in either case, different bacteria in the strain cannot ] be screened. Thus, the experimental results can only be reflected in the flora, which do | not verify the specific strains. This leads to some accuracy problems in the experimental | results. | SUMMARY OF INVENTION ;
[0009] The object of the present invention is to provide a preliminary screening method of bacteria having an ammonia nitrogen degradation function, different bacterial species can : be distinguished based on the ammonia nitrogen degradation rate in the experiment, and which provides a reliable guarantee for the test results of the subsequent ammonia nitrogen degradation of bacteria to solve the problems mentioned in the above background.
[0010] To achieve the foregoing objective, the present invention provides the following technical solutions:
[0011] A preliminary screening method of bacteria having an ammonia nitrogen degradation function comprising the following steps:
[0012] S1: preparing an ammonia nitrogen selective culture medium by using materials including (NH4)2S04: 1.18 g/L; FeSO4 7H20: 0.04 g/L; KoHPO4: 1 g/L; KHaPO4: 0.25 g/L; MgSO4-7TH20: 0.5 g/L; glucose: 10 g/L; trace elements solution: 1 mL; : |
[0013] S2: preparing an ammonia nitrogen standard stock solution, pN=1000 pg/mL; weighing 3.819 g ammonium chloride, then drying the ammonium chloride in an oven and | dissolving the ammonium chloride in water, and then transferring the ammonium chloride | solution into a 1000 mL volumetric flask and diluting to the mark line; |
[0014] 83: preparing an ammonia nitrogen standard working solution, pN=10 pg/mL, | pipetting 5.00 mL of the ammonia nitrogen standard stock solution into a 500 mL | volumetric flask and then diluting to the mark; |
[0015] S4: adding 0.00, 0,10, 0.20, 0.40, 0.80, 1.20, 1.60 and 2.00 mL of the ammonia | nitrogen standard working solution into eight 10 mL colorimetric tubes respectively, | adding water to the mark line, then adding 200 mL of a potassium sodium tartrate solution and shaking well, then adding 300 mL of Nessler’s reagent and shaking well, and then | placing the colorimetric tubes for 10 min and measuring absorbance by using a 10 mm | cuvette at a wavelength of 420 nm with water as a reference; |
[0016] 85: inoculating strains: pipetting 1 mL of a bacteria liquid into a 1.5 mL sterile | centrifuge tube and centrifuging at 5000 rpm for 5 minutes, discarding the supernatant, resuspending the bacteria with sterile water, repeating three times to wash away the residual ’ medium on the bacteria, and inoculating 10% of the bacteria into the selective culture medium, and setting up a control group inoculated with sterile water at the same tims, culturing for 3 days and 6 days at 180 rpm and 34.5 °C; 35 [0017] S86: detecting the ammonia nitrogen content of the ammonia nitrogen selective | culture medium by using a Nessler's reagent spectrophotometry method, the ammonia | nitrogen degradation rate = (the ammonia nitrogen content in a control group — the ammonia nitrogen content in an experimental group) / the ammonia nitrogen content in the | control group. :
[0018] Further, the trace elements solution of the ammonia nitrogen selective culture medium in 81 is prepared by using materials including zinc sulfate heptahydrate 3.9 g, calcium # chloride dihydrate 7 g, manganese chloride tetrahydrate 5.1 g, ammonium molybdate 1.1 | g, copper sulfate pentahydrate 1.6 g, cobalt chloride hexahydrate 1.6 g, distilled water 1000 | ml. |
[0019] Compared with prior arts, beneficial effects of the present invention are as follow: | [00201 The preliminary screening method of bacteria having an ammonia nitrogen 1 degradation function provided herein can screen the bacteria with different ammonia 9 nitrogen degradation functions out in microbial bacteria, this is not available in the existing | experimental methods, so that a favorable guarantee is provided for a subsequent bacteria | differentiate experiment, the gap of existing experimental screening of bacteria having an ammonia nitrogen degradation function is filled, and the ammonia nitrogen degradation } experimental method is further improved. DESCRIPTION OF DRAWINGS |
[0021] Figure 1 is the standard curve diagram of the ammonia nitrogen content of the present invention. | |
[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the | 55 present invention,
[0023] The embodiment of the invention provides a preliminary screening method of bacteria | having an ammonia nitrogen degradation function comprising the following steps:
[0024] Step 1: preparing an ammonia nitrogen selective culture medium by using materials | including (NH4)250:: 1.18 g/L; FeS04- TH20: 0.04 g/L; K2HPO:: 1 g/L; KH2PO4: 0.25 g/L; MgS04-TH20: 0.5 g/L; glucose: 10 g/L; trace elements solution: 1 mL; as shown in Table
1 below: | Table 1: Components of the ammonia nitrogen selective medium | Reagent name Amount | (NH4)2504 1.18 g/L. | Fe5O4 7H20 0.04 g/L | KoHPO, 1 g/l | KH2PO4 0.25 g/L | Mgs047H20 0.5 g/L. Glucose 10 g/L Trace elements solution 1 mL
[0025] Wherein the trace elements solution including zine sulfate heptahydrate 3,9 g, calcium chloride dihydrate 7 g, manganese chloride tetrahydrate 5.1 g, ammonium molybdate 1.1 | g, copper sulfate pentahydrate 1.6 g, cobalt chloride hexahydrate 1.6 g, distilled water 1000 | ml. |
[0026] Step 2: preparing an ammonia nitrogen standard stock solution, pN=1000 pg/ml; weighing 3.819 g ammonium chloride, then drying the ammonium chloride in an oven and | dissolving the ammonium chloride in water, and then transferring the ammonium chloride | solution into a 1000 mL volumetric flask and diluting to the mark line;
[0027] Step 3: preparing an ammonia nitrogen standard working solution, pN=10 pg/mL, | pipetting 5.00 ml of the ammonia nitrogen standard stock solution into a 500 mL | volumetric flask and then diluting to the mark; |
[0028] Step 4: adding 0.00, 0.10, 0.20, 0.40, 0.80, 1.20, 1.60 and 2.00 mL of the ammonia | nitrogen standard working solution into eight 10 mL colorimetric tubes respectively, adding water to the mark line, then adding 200 mL of a potassium sodium tartrate solution and shaking well, then adding 300 mL of Nessler's reagent and shaking well, and then placing the colorimetric tubes for 10 min and measuring absorbance by using a 10 mm cuvette at a wavelength of 420 nm with water as a reference;
[0029] Step 5: inoculating strains: pipetting 1 mL of a bacteria liquid into a 1.5 mL sterile | centrifuge tube and centrifuging at 5000 rpm for 5 minutes, discarding the supernatant,
resuspending the bacteria with sterile water, repeating three times to wash away the residual medium on the bacteria, and inoculating 10% of the bacteria into the selective culture mediurn, and setting up a control group inoculated with sterile water at the same time, | culturing for 3 days and 6 days at 180 rpm and 34.5 °C; |
[0030] Step 6: detecting the ammonia nitrogen content of the ammonia nitrogen selective culture medium by using a Nessler's reagent spectrophotometry method, the ammonia | nitrogen degradation rate = (the ammonia nitrogen content in a control group — the 5 ammonia nitrogen content in an experimental group) / the ammonia nitrogen content in the # control group.
[0031] Results: |
[0032] Please refer to Figure 1, the results of the standard curve of the ammonia nitrogen | content; |
[0033] Refer to Table 2 below, the screening results of the ammonia nitrogen degradation | bacteria: | Table 2: Ammonia nitrogen degradation rate index results | Ammonia nitrogen No Latin name Source degradation rate : 1 Bacillus subtiliis Complex bacteria 47.16% 9 2 Bacillus subtilis Complex bacteria 46.28% | 3 Bacillus subtiliis Deodorant bacteria ~~ 45.84% | 4 Bacillus subtiliis River water 47.16% 9 5 Bacillus subtilis Dajianshan tree farm 44.07% î Air raid shelter water 6 Bacillus subtiliis 53.33% sample Bacillus velezensis î 10 44.29% Bacillus siamensis 14 Bacillus velezensis 43.19% |
7 | | Bacillus Environment : | 50 53.00% amyloliquefaciens ; Klebsiella | 7 Human 53.77% quasipneumoniae : Klebsiella | 8 Eclectus Parrot 54.65% pneumoniae | 24 Klebsiella variicola Human 54,15% | Klebsiella 25 Red wombat 52.09% pneumoniae Klebsiella ‘ | 26 Human 52.09% pneumoniae Klebsiella | 39 Human 47.05% pneumoniae | Klebsiella Black-collared | 40 52.01% : pneumoniae strain Starling | Klebsiella | 41 Psittacula alexandri 35.66% pneumoniae strain Klebsiella | 42 Human 47.55% pneumoniae : 43 Klebsiella variicola ~~ Human 16.35% 44 Klebsiella variicola ~~ Human 51.51% 45 Klebsiella variicola ~~ Human 48.54% 9 Bacillus megaterium Anteater 13.66% 11 Bacillus megaterium 4.37% 12 Bacillus megaterium 13.12% 27 Bacillus megaterium Dajianshan tree farm 2.99% 28 Bacillus megaterium Roof flowerpot 2.49% :
Bacillus | Farmland soil | 29 aryabhattai/Bacillus 7.96% samples | megaterium ; Bacillus | Flowerbed soil | 30 aryabhattai/Bacillus 7.47% : samples ‘ | megaterium . 33 Bacillus megaterium Mallard greylag 846% | 34 Bacillus megaterium Panther 1.99% 35 Bacillusmegaterium Conghua Citrus field 0.50% 36 Bacillus megaterium Conghua Citrus field -0.50% Environmental : 37 Bacillus megaterium 4.48% samples 38 Bacillus megaterium Hippopotamus -3.98% Bacillus | 15 42.65% | licheniformis Bacillus | 46 River water 29.72% licheniformis Complex microbial : Bacillus | 47 agent of yellow 2.48% licheniformis shoot Complex microbial Bacillus 48 agent of yellow 22.78% licheniformis shoot Bacillus : 49 Gnu 39.13% licheniformis Bacillus Environmental 51 35.66% : licheniformis samples : 13 Brevibacillus 3.83% mee parabrevis | 16 Bacillus cereus Tiger zone -1.09% 18 Bacillus toyonensis Gorilla Pavilion -1.00% | 19 Bacillus cereus Wolf zone -3.62% | 21 Bacillus Kangaroo Pavilion 2.27% | 22 Bacillus cereus Anteater -8.61% 23 Bacillus altitudinis Tiger zone 34.04% Acinetobacter | 52 Manis javanica 16.46% baumannii : Acinetobacter 53 Human 21.35% baumannii Acinetobacter 54 Tapirus bairdii 10.23% baumannii
Acinetobacter : 55 Psittacus erithacus 42.26% baumannii strain Air raid shelter water 56 Acinetobacter junii 52.50% sample 57 Acinetobacter junii ~~ Erythrocebus patas ~~ 25.80% Acinetobacter 58 tandoii/Acinetobacter River water 60.06% junii 59 Acinetobacter sp.
Conghua Citrus field 59.61% Acinetobacter Environmental 60 13.79% nosocomialis samples 61 Acinetobacter tandoit Pyrrhura molinae 56.94% Macropus 62 Acinetobacter sp. 4.89% rufogriseus
63 Acinetobacter sp.
Pony 3.11% 64 Acinetobacter lwoffii Boar 4.89% | 65 Acinetobacter lwoffii Eland 445% à Acinetobacter ; 66 Panther -1.78% | calcoaceticus Acinetobacter 67 Panda 1.78% calcoaceticus à Acinetobacter 68 Sturgeon 52.94% | calcoaceticus
Acinetobacter : 69 River water 2.67% johnsonii 70 Enterobacter cloacae Beasty Birds 10.23% ] 71 Enterobacter cloacae Parus spilonotus 48.05% 9 72 Enterobacter cloacae Derbyan Parakeet 4,00% | 73 Enterobacter cloacae Scarlet macaw 53.39% | Streptopelia 74 Proteus hauseri 36.23% orientalis 50 75 Proteus hauseri Rhinoceros 28.08% | 76 Proteus mirabilis Paguma larvata 23.17% | 77 Proteus mirabilis Manis javanica 0,45% | 78 Proteus mirabilis Panda 37.50% 79 Proteus mirabilis Saimiri sciureus 2 35.78%
Proteus vulgaris Chiorocebus sabaeus 80 4.98% : Proteus vulgaris 1 81 Proteus mirabilis Caracal caracal 51.63% 82 Proteus mirabilis Human -1.81%
11 | Panthera tigris | 83 Proteus mirabilis 55.26% Amoyensis 84 Proteus mirabilis Cacatua galerita 52.54% |
[0034] Analysis of the experimental results: | > [0035] The ammonia nitrogen degradation rates of 9 strains of Bacillus subtilis (including | Bacillus velezensis and Bacillus amyloliquefaciens) were 35.66% - 53.33%, the ammonia | nitrogen degradation rates of 12 strains of Klebsiella were 36.66% - 54.65%, the ammonia # nitrogen degradation rates of 13 strains of Bacillus megaterium were 2.86% - 14.77%, the | ammonia nitrogen degradation rates of 4 strains of Bacillus cereus were 0%, the ammonia | 0 nitrogen degradation rates of 6 strains of Bacillus licheniformis were 2.48% - 42.65%, the | ammonia nitrogen degradation rates of 18 strains of Acinetobacter were 0%-60.06%, the | ammonia nitrogen degradation rates of 4 strains of Enterobacter cloacae were 4.0%: - |
53.39%, and the ammonia nitrogen degradation rates of 11 strains of Proteus mirabilis were 0% - 55.26%. | to [0036] In conclusion, the preliminary screening method of bacteria having an ammonia nitrogen degradation function provided herein can screen the bacteria with different ammonia nitrogen degradation functions out in microbial bacteria, so that a favorable guarantee is provided for a subsequent bacteria differentiate experiment, the gap of existing experimental screening of bacteria having an ammonia nitrogen degradation function is | filled, and the ammonia nitrogen degradation experimental method is further improved. |
[0037] The above are only preferred specific embodiments of the present invention, but the scope of protection of the present invention is not limited to this. Equivalent replacements | or changes made by anyone familiar with the technical field within the technical scope | disclosed in the present invention according to the technical solution and its inventive | 2 concept of the present invention should all fall within the protection scope of the present | invention, | |
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