CN109234188B - Heterotrophic nitrification aerobic denitrifying bacterium L2 and application thereof - Google Patents
Heterotrophic nitrification aerobic denitrifying bacterium L2 and application thereof Download PDFInfo
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Abstract
The invention discloses a heterotrophic nitrification aerobic denitrifying bacterium L2 and application thereof, wherein the strain is Xenophilus sp.L2, and is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms, and the registration number of the preservation center is as follows: CGMCC No. 15918. The bacterium can carry out heterotrophic nitrification and/or aerobic denitrification, and can be applied to the treatment and restoration of nitrogen-polluted water bodies.
Description
Technical Field
The invention belongs to the field of environmental microorganisms, and particularly relates to a heterotrophic nitrification aerobic denitrifying bacterium L2 and application thereof.
Background
The pollution problem of nitrogen to the environment is increasingly prominent in recent years, and the harmfulness of nitrogen is increasingly recognized and valued. Such as ammonia nitrogen, nitrate nitrogen and nitrite nitrogen, are potentially converted into carcinogenic, mutagenic and teratogenic nitrosamines; and nitrogen flows into the water body to cause eutrophication of the water body, which causes deterioration of water quality and even deterioration of lakes. The biological denitrification has the advantages of good treatment effect, stable and reliable treatment process, convenient operation and management and the like, and is widely applied.
The traditional nitrifying bacteria are autotrophic bacteria, but the growth environment is strict, the growth speed is slow, and the application is limited due to the difficulty in mass culture. The heterotrophic nitrifying bacteria can directly utilize organic matter nutrition in the water body to grow and degrade ammonia nitrogen, the growth speed is high, and COD and ammonia nitrogen can be removed simultaneously. Meanwhile, denitrification is generally considered to be carried out under anaerobic condition, but some heterotrophic nitrifying bacteria can carry out aerobic denitrification, which has great significance for removing nitrogen pollutants.
Disclosure of Invention
The invention aims to provide a heterotrophic nitrification aerobic denitrifying bacterium L2 and application thereof.
The invention adopts the specific scheme that:
a heterotrophic nitrification aerobic denitrification bacterium L2 is Xenophilus sp.L2, is preserved in the China general microbiological culture Collection center, and has the registration number of: CGMCC No. 15918.
The invention also provides application of the heterotrophic nitrification aerobic denitrifying bacteria L2, wherein the heterotrophic nitrification aerobic denitrifying bacteria L2 are inoculated in a nitrogen-polluted water body for heterotrophic nitrification and/or aerobic denitrification.
The invention has the following beneficial effects:
the heterotrophic nitrification aerobic denitrification bacteria L2 can synchronously realize heterotrophic nitrification and aerobic denitrification, and has great economic value.
Drawings
FIG. 1 is an electrophoresis diagram of a PCR product of heterotrophic nitrification-aerobic denitrifying bacteria L2;
FIG. 2 is a phylogenetic tree of strain L2 and the 16S rDNA sequence of related species.
Strain preservation
The invention relates to Xenophilus sp.L2, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation address is as follows: west road No.1, north west of chaoyang district, beijing, registration number of preservation center is CGMCC No.15918, preservation date: 6 and 8 months in 2018.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.
Example 1: separation of heterotrophic nitrification aerobic denitrifying bacteria L2
Taking 5g of river sediment in Tianjin Jizhou region, adding 100ml of enrichment medium (NH) sterilized at 121 DEG C4Cl 0.382g/L、KNO30.36g/L, sodium acetate 2g/L, MgSO4·7H2O 0.2g/L、K2HPO4 0.2g/L、NaCI 0.12g/L、MnSO4·4H2O 0.01g/L、FeSO40.01g/L, water in balance, pH 7.0-7.2), at 30 ℃ and 1The aerobic culture was carried out at 50rpm for 3 days with shaking, and then the culture was carried out at 30 ℃ for 3 days with standing, and this was repeated once more. Then 5ml of the enriched liquid is taken and inoculated into 100ml of enrichment culture medium (NH) sterilized at 121 DEG C4Cl 0.382g/L、KNO30.36g/L, sodium acetate 2g/L, MgSO4·7H2O 0.2g/L、K2HPO4 0.2g/L、NaCI 0.12g/L、MnSO4·4H2O 0.01g/L、FeSO40.01g/L, water in balance, pH 7.0-7.2), aerobically culturing at 30 deg.C and 150rpm for 3d, and then culturing at 30 deg.C and standing for 3d, and repeating the above steps. Then taking the enriched liquid obtained by secondary enrichment and using a solid enrichment culture medium (agar 15g/L, NH)4Cl0.382g/L、KNO30.36g/L, sodium acetate 2g/L, MgSO4·7H2O 0.2g/L、K2HPO4 0.2g/L、NaCI 0.12g/L、MnSO4·4H2O 0.01g/L、FeSO40.01g/L and the balance of water, pH 7.0-7.2), and then carrying out dilution coating, picking single colonies, and separating to obtain a strain L2.
Example 2 identification of heterotrophic nitrification-aerobic denitrifying bacteria L2
Morphological characteristics of L2: short rods, no spores were produced, and cyan colonies formed on solid plates. Physiological and biochemical characteristics of L2: gram staining is negative, urease reaction is negative. Activated L2, CGMCC No.15918 was inoculated in liquid LB medium (10g/L peptone, 5g/L yeast powder, 10g/L NaCl) and cultured with shaking at 150rpm at 30 ℃ for 24 hours. Centrifuging 1ml of fresh culture solution at 4 ℃ and 8000rpm for 5min, collecting thallus, extracting DNA by using a DNA extraction kit, performing PCR amplification by using a universal primer 8F/1492R, detecting electrophoresis of PCR products, determining an electrophoresis band shown as a lane 3 in figure 1, determining a 16S rDNA gene sequence (shown as SEQ ID NO: 1), and determining that L2 belongs to Xenophilus sp. The phylogenetic tree of the 16S rDNA sequence of strain L2 and related species is shown in FIG. 2.
A sequence table: 1, SEQ ID NO:
ttcggatgctgacgagtggcgaacgggtgagtaatacatcggaacgtgcccgatcgtgggggatagctcggcgaaagccggattaataccgcatacgatctacggatgaaagcaggggaccgcaaggccttgcgcgaacggagcggccgatggcagattaggtagttggtggggtaaaggctcaccaagccgacgatctgtagctggtctgagaggacgaccagccacactgggactgagacacggcccagactcctacgggaggcagcagtggggaattttggacaatgggcgcaagcctgatccagcaatgccgcgtgcaggatgaaggccttcgggttgtaaactgcttttgtacggaacgaaaaggctctggttaatacctggggctcatgacggtaccgtaagaataagcaccggctaactacgtgccagcagccgcggtaatacgtagggtgcgagcgttaatcggaattactgggcgtaaagcgtgcgcaggcggtgatgtaagacagttgtgaaatccccgggctcaacctgggaactgcatctgtgactgcatcgctggagtgcggcagagggggatggaattccgcgtgtagcagtgaaatgcgtagatatgcggaggaacaccgatggcgaaggcaatcccctgggcctgcactgacgctcatgcacgaaagcgtggggagcaaacaggattagataccctggtagtccacgccctaaacgatgtcaactggttgttgggaatttgctttctcagtaacgaagctaacgcgtgaagttgaccgcctggggagtacggccgcaaggttgaaactcaaaggaattgacggggacccgcacaagcggtggatgatgtggtttaattcgatgcaacgcgaaaaaccttacccacctttgacatgtacggaactcgccagagatggcttggtgctcgaaagagagccgtaacacaggtgctgcatggctgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgtcattagttgctacattcagttgggcactctaatgagactgccggtgacaaaccggaggaaggtggggatgacgtcaagtcctcatggcccttataggtggggctacacacgtcatacaatggctggtacaaagggttgccaacccgcgagggggagctaatcccataaagccagtcgtagtccggatcgcagtctgcaactcgactgcgtgaagtcggaatcgctagtaatcgtggatcagaatgtcacggtgaatacgttcccgggtcttgtacacaccgcccgtcacaccatgggagcgggttctgccagaagtagttag
(2) l2, CGMCC No.15918 heterotrophic nitrification function detection
Activated L2, CGMCC No.15918 was inoculated in liquid LB medium (10g/L peptone, 5g/L yeast powder, 10g/L sodium chloride), and cultured with shaking at 150rpm at 30 ℃ for 24 hours. Inoculating 5% (V/V) fresh bacterial liquid into heterotrophic nitrification simulation wastewater (NH)4Cl 0.382g/L, sodium acetate 2g/L, MgSO4·7H2O 0.2g/L、K2HPO4 0.2g/L、NaCI 0.12g/L、MnSO4·4H2O 0.01g/L、FeSO40.01g/L, pH7.0-7.2), and culturing aerobically at 30 deg.C and 150rpm for 7 d. There was a blank control without inoculation. After 7 days of incubation, the presence of nitrite or nitrate in the broth was checked using grits' reagent and diphenylamine reagent. If nitrite or nitrate exists in the culture solution, the heterotrophic nitrification is indicated to be positive; if nitrite and nitrate are not present in the culture solution, no occurrence of the difference is indicatedNutrition and nitrification are negative for heterotrophic nitrification.
The detection results of the Grignard reagent and the diphenylamine reagent show that the L2 and CGMCC No.15918 have positive heterotrophic nitrification and have the function of heterotrophic nitrification.
The relationship between different concentrations (mg/L) of ammonia nitrogen and OD values is established according to a standard HJ 535-2009 reagent spectrophotometry for measuring ammonia nitrogen in water, and the relationship is shown in table 1, wherein the OD value is taken as a horizontal coordinate, the ammonia nitrogen concentration is taken as a vertical coordinate, a standard curve is drawn, and a curve formula is as follows: y 5.3087x +0.0005, wherein R2＝0.9996。
TABLE 1 Ammonia nitrogen and OD values at different concentrations (mg/L)
| NH3-N | 0 | 0.1 | 0.2 | 0.6 | 1 | 1.2 | 1.4 | 1.6 | 2 |
| OD value | 0.007 | 0.026 | 0.043 | 0.12 | 0.195 | 0.233 | 0.272 | 0.313 | 0.379 |
Similarly, according to the standard HJ 535-2009 ammonia nitrogen determination naeskins reagent spectrophotometry, the solutions cultured in the blank control group and the L2 experimental group are diluted by 50 times (so that the ammonia nitrogen concentration is 0.1-2mg/L) to determine the OD value of the diluent (see table 2), the ammonia nitrogen concentration in the diluent is calculated according to the standard curve formula y of 5.3087x +0.0005 after correction (see table 2), and the ammonia nitrogen concentration in the original solution cultured in the blank control group and the L2 experimental group is determined according to the dilution factor (see table 2).
TABLE 2 ammonia nitrogen determination for blank control and L2 test groups
Calculating the ammonia nitrogen degradation rate of L2 according to the ammonia nitrogen concentration of the stock solution of the blank control group-the ammonia nitrogen concentration of the stock solution of the L2 experiment group)/the ammonia nitrogen concentration of the stock solution of the blank control group, wherein the ammonia nitrogen degradation rate of CGMCC No.15918 heterotrophic nitrification is (95.05-44.09)/95.05 is 53.61%.
Example 4 detection of aerobic Denitrification function in L2 CGMCC No.15918
Activated L2, CGMCC No.15918 was inoculated in liquid LB medium (10g/L peptone, 5g/L yeast powder, 10g/L sodium chloride), and cultured with shaking at 150rpm at 30 ℃ for 24 hours. Inoculating 5% (V/V) fresh bacteria liquid into aerobic denitrification simulated wastewater (KNO)30.36g/L, sodium acetate 2g/L, MgSO4·7H2O 0.2g/L、K2HPO4 0.2g/L、NaCI 0.12g/L、MnSO4·4H2O 0.01g/L、FeSO40.01g/L, pH 7.0-7.2), 30 ℃ and 150rpm shakingAnd (7) carrying out aerobic culture for 7 d. There was a blank control without inoculation. After 7 days of incubation, the presence of nitrite and depletion of nitrate in the broth was checked using grignard and diphenylamine reagents. If nitrite exists in the culture solution, the aerobic denitrification is performed, and the culture solution is positive; if the nitrate in the culture solution is consumed, the aerobic denitrification is also shown to occur, and the aerobic denitrification is also positive.
The detection results of the Grignard reagent and the diphenylamine reagent show that the L2 and CGMCC No.15918 are aerobic and denitrification positive and have the aerobic denitrification function.
Establishing a relation between different concentrations (mg/L) and OD values of total nitrogen according to a standard HJ 636-: y 18.724x +0.2922 wherein R2＝0.9993。
TABLE 3 Total Nitrogen and OD values at different concentrations (mg/L)
| Total nitrogen | 0 | 0.5 | 1 | 2.5 | 5 | 6 |
| A220 | 0.0638 | 0.0794 | 0.104 | 0.1848 | 0.3112 | 0.3767 |
| A275 | 0.0051 | 0.0064 | 0.0075 | 0.0056 | 0.0055 | 0.0075 |
| A220-2A275 | 0.0536 | 0.0666 | 0.089 | 0.1736 | 0.3002 | 0.3617 |
Similarly, according to the alkaline potassium persulfate digestion ultraviolet spectrophotometry for measuring total nitrogen in the standard HJ 636-.
TABLE 4 Total Nitrogen determination for blank control and L2 test groups
And (3) calculating the total nitrogen degradation rate of L2, CGMCC No.15918 and aerobic denitrification (51.01-20.02)/51.01 (60.74%) according to the total nitrogen concentration of the stock solution of the blank control group-L2 experiment group/the total nitrogen concentration of the stock solution of the blank control group.
Sequence listing
<110> Lixiaoming
<120> heterotrophic nitrification aerobic denitrifying bacterium L2 and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1355
<212> DNA
<213> xenobiotic bacterium (Xenophilus sp.)
<400> 1
ttcggatgct gacgagtggc gaacgggtga gtaatacatc ggaacgtgcc cgatcgtggg 60
ggatagctcg gcgaaagccg gattaatacc gcatacgatc tacggatgaa agcaggggac 120
cgcaaggcct tgcgcgaacg gagcggccga tggcagatta ggtagttggt ggggtaaagg 180
ctcaccaagc cgacgatctg tagctggtct gagaggacga ccagccacac tgggactgag 240
acacggccca gactcctacg ggaggcagca gtggggaatt ttggacaatg ggcgcaagcc 300
tgatccagca atgccgcgtg caggatgaag gccttcgggt tgtaaactgc ttttgtacgg 360
aacgaaaagg ctctggttaa tacctggggc tcatgacggt accgtaagaa taagcaccgg 420
ctaactacgt gccagcagcc gcggtaatac gtagggtgcg agcgttaatc ggaattactg 480
ggcgtaaagc gtgcgcaggc ggtgatgtaa gacagttgtg aaatccccgg gctcaacctg 540
ggaactgcat ctgtgactgc atcgctggag tgcggcagag ggggatggaa ttccgcgtgt 600
agcagtgaaa tgcgtagata tgcggaggaa caccgatggc gaaggcaatc ccctgggcct 660
gcactgacgc tcatgcacga aagcgtgggg agcaaacagg attagatacc ctggtagtcc 720
acgccctaaa cgatgtcaac tggttgttgg gaatttgctt tctcagtaac gaagctaacg 780
cgtgaagttg accgcctggg gagtacggcc gcaaggttga aactcaaagg aattgacggg 840
gacccgcaca agcggtggat gatgtggttt aattcgatgc aacgcgaaaa accttaccca 900
cctttgacat gtacggaact cgccagagat ggcttggtgc tcgaaagaga gccgtaacac 960
aggtgctgca tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1020
gcgcaaccct tgtcattagt tgctacattc agttgggcac tctaatgaga ctgccggtga 1080
caaaccggag gaaggtgggg atgacgtcaa gtcctcatgg cccttatagg tggggctaca 1140
cacgtcatac aatggctggt acaaagggtt gccaacccgc gagggggagc taatcccata 1200
aagccagtcg tagtccggat cgcagtctgc aactcgactg cgtgaagtcg gaatcgctag 1260
taatcgtgga tcagaatgtc acggtgaata cgttcccggg tcttgtacac accgcccgtc 1320
acaccatggg agcgggttct gccagaagta gttag 1355
Claims (2)
1. A heterotrophic nitrification aerobic denitrification bacterium L2 is characterized in that: the strain is Xenophilus sp.L2, is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms, and has the registration number of: CGMCC No. 15918.
2. The use of the heterotrophic nitrification-aerobic denitrifying bacteria L2 as claimed in claim 1, wherein: and inoculating the heterotrophic nitrification aerobic denitrification bacteria L2 into a nitrogen-polluted water body for heterotrophic nitrification and/or aerobic denitrification.
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| CN101351559A (en) * | 2005-11-09 | 2009-01-21 | 普里梅拉生物系统有限公司 | Multiplexed quantitative detection of pathogens |
| CN105950514A (en) * | 2016-06-08 | 2016-09-21 | 北京润世科技有限公司 | Microbial oil recovery bacteria W-Y3 and application thereof |
| BE1026952A1 (en) * | 2018-12-26 | 2020-07-30 | Avecom Nv | Method for converting starch-containing residual flows into high-quality proteins |
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| SBＲ 系统外加磁场对微生物群落多样性和处理效果;耿淑英等;《环境科学》;20171130;第38卷(第11期);第4715-4724页 * |
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