CN102586132B - Sphingobacteria sp. for removing ammonia nitrogen from sewage at low temperature and separate culturing method thereof - Google Patents
Sphingobacteria sp. for removing ammonia nitrogen from sewage at low temperature and separate culturing method thereof Download PDFInfo
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- CN102586132B CN102586132B CN2011104169299A CN201110416929A CN102586132B CN 102586132 B CN102586132 B CN 102586132B CN 2011104169299 A CN2011104169299 A CN 2011104169299A CN 201110416929 A CN201110416929 A CN 201110416929A CN 102586132 B CN102586132 B CN 102586132B
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Abstract
The invention discloses sphingobacteria sp. for removing ammonia nitrogen from sewage at a low temperature and a separate culturing method thereof. The sphingobacteria sp. sub-white 1CGMCC No.5278 for removing ammonia nitrogen from sewage at a low temperature has the capability of removing ammonia nitrogen from sewage under a low-temperature aerobic condition. As proved by an experimental result, the sphingobacteria sp. for removing ammonia nitrogen from sewage at a low temperature disclosed by the invention has remarkable strengthening effect on the removal of ammonia nitrogen from sewage at a low temperature, particularly under the condition of 8-12 DEG C even though being remarkably suppressed by a low-temperature environment. Compared with sewage into which sphingobacteria sp. is not inoculated, sewage into which sphingobacteria sp. is inoculated has the advantages that: the removing rate of ammonia nitrogen is increased by about 10 percent, and the concentration of effluent ammonia nitrogen is 5.0 mg/L in maximum, being superior to a grade A discharge standard (when the water temperature is lower than or equal to 12 DEG C, the control index of the grade A discharge standard is 8 mg/L).
Description
Technical field
The invention belongs to sewage treatment area, relate to a strain and have low temperature and go down except sphingolipid bacillus and the isolation cultivation method of ammonia nitrogen in the sewage.
Background technology
Ammonia nitrogen is one of principal pollutant that cause body eutrophication.At present, removing ammonia nitrogen mainly is to finish by the autotrophy nitrobacteria, but winter low temperature has stronger restraining effect to nitrobacteria.Mainly be because the reduction of temperature has influenced microbial growth and metabolism and made that change has taken place microbial population in the Sewage treatment systems.In temperature decline process, the metabolic activity of mesophilic bacteria descends gradually, just lose activity during to certain temperature, and be suitable for low temperature environment psychrophile quantity since self physiological property and the restraining effect of various ecological factors, quantitatively can't reach the status of dominant microflora, thereby cause sewage work's effluent quality in winter to descend.Therefore need badly a kind of can be in the low-temperature aerobic condition be gone down except sewage the bacterial strain of ammonia nitrogen.
Summary of the invention
The objective of the invention is to overcome existing bacterial strain low temperature ammonia nitrogen removal poor effect, a kind of sphingolipid bacillus that can remove ammonia nitrogen in the sewage at low temperatures is provided.
Second purpose of the present invention provides a kind of low temperature and goes down except the isolation cultivation method of the sphingolipid bacillus of ammonia nitrogen in the sewage.
The 3rd purpose of the present invention provides a kind of low temperature and goes down except the purposes of the sphingolipid bacillus of ammonia nitrogen in the sewage.
Technical scheme of the present invention is summarized as follows:
A kind of low temperature goes down except the inferior white 1CGMCCNo.5278 of the sphingolipid bacillus of ammonia nitrogen in the sewage (Sphingobacteria sp.), and it has the ability of ammonia nitrogen in the low-temperature aerobic condition is gone down except sewage.
A kind of low temperature goes down except the purposes of the inferior white 1CGMCC No.5278 ammonia nitrogen in the low-temperature aerobic condition is gone down except sewage of the sphingolipid bacillus of ammonia nitrogen in the sewage (Sphingobacteria sp.).
Described low temperature is preferably 8-12 ℃.
Low temperature goes down except the isolation cultivation method of the sphingolipid bacillus of ammonia nitrogen in the sewage, comprises the steps:
(1) first kind of Nitrite bacteria nutrient solution put into lab scale sbr reactor device, sludge of sewage treatment plant is inoculated in described first kind of Nitrite bacteria nutrient solution, at 8～12 ℃, cultured continuously 4-6 days, take out supernatant liquor, add second kind of Nitrite bacteria nutrient solution, at 8～12 ℃, cultured continuously 4-6 days, take out supernatant liquor, add the third Nitrite bacteria nutrient solution, at 8～12 ℃, cultured continuously 4-6 days, enrichment, domestication psychrotropic bacteria;
(2) get the mud that 10mL step (1) obtains, add oneself and be equipped with in the 250mL Erlenmeyer flask of 90mL sterile purified water and 20～30 sterilization granulated glass spherees, vibration 10～30min makes bacterial cell fully be discharged in the supernatant liquor; Leave standstill 20～40min, get the adding of 1mL supernatant liquor and be equipped with in the test tube of 9mL sterile distilled water, obtain 10
-2Bacteria suspension, take turns doing serial dilution as stated above, accomplish 10 always
-8Till;
(3) choosing gradient is 10
-5～10
-8Bacteria suspension, get 1mL respectively and be inoculated in the Nitrite bacteria solid medium, each extent of dilution is made 3 parallel samples, the numbering; Under the condition of aerobic, preserve moisture for 10 ℃ and cultivated for 2～3 weeks;
(4) choosing colony is big in the Nitrite bacteria solid medium flat board from step (3), single bacterium colony that form is different, line separates again in Nitrite bacteria solid medium flat board, repeating plate streaking separates 3-4 time, make the homomorphosis of bacterium colony in microscope on the flat board, single, big or small similar single bacterium colony that is, called after is inferior white 1, through be accredited as cold condition go down except sewage in the sphingolipid bacillus (Sphingobacteria sp.) of ammonia nitrogen
Described first kind of Nitrite bacteria nutrient solution is made up of following component: NH
4Cl 0.0764g, sodium acetate 2g, MgSO
47H
2O0.05g, K
2HPO
40.2g, NaCl 0.12g, MnSO
4H
2O 0.01g, FeSO
40.01g adding distil water is to 1000mL.
Described second kind of Nitrite bacteria nutrient solution is made up of following component: NH
4Cl 0.191g, sodium acetate 2g, MgSO
47H
2O0.05g, K
2HPO
40.2g, NaCl 0.12g, MnSO
4H
2O 0.01g, FeSO
40.01g adding distil water is to 1000mL.
Described the third Nitrite bacteria nutrient solution is made up of following component: NH
4Cl 0.382g, sodium acetate 2g, MgSO
47H
2O0.05g, K
2HPO
40.2g, NaCl 0.12g, MnSO
4H
2O 0.01g, FeSO
40.01g adding distil water is to 1000mL.
Described Nitrite bacteria solid medium is made up of following component: NH
4Cl 0.0764g, sodium acetate 2g, MgSO
47H
2O0.05g, K
2HPO
40.2g, NaCl 0.12g, MnSO
4H
2O 0.01g, FeSO
40.01g, agar 20g, adding distil water is to 1000mL.
Experimental result shows: although be subjected to the obvious inhibition of low temperature environment, adding a kind of low temperature of the present invention goes down except the inferior white 1CGMCC No.5278 of the sphingolipid bacillus (Sphingobacteria sp.) of ammonia nitrogen in the sewage, at low temperatures, particularly under 8-12 ℃ condition, ammonia nitrogen removal in the sewage there is tangible strengthening effect, inoculating sphingolipid bacillus of the present invention compares with the sewage of not inoculating bacterium, ammonia nitrogen removal frank has improved and has been about 10%, the water outlet ammonia nitrogen concentration is up to 5.0mg/L, what be better than one-level A goes out water quality standard (water temperature≤12 ℃ time, the control index of one-level A standard is 8mg/L).
Description of drawings
Fig. 1 goes down except a kind of domestication separation and Culture schema of removing the sphingolipid bacillus of ammonia nitrogen in the sewage of ammonia nitrogen in the sewage for the low-temperature aerobic condition.
Fig. 2 is Ya Bai-1 ammonia nitrogen removal test chart.
Fig. 3 is Ya Bai-1 method for determining bacteria flow process.
Embodiment
The present invention is further illustrated below by specific embodiment.
Embodiment 1
Low temperature goes down except the isolation cultivation method of the sphingolipid bacillus of ammonia nitrogen in the sewage, comprises the steps:
(1) first kind of Nitrite bacteria nutrient solution put into lab scale sbr reactor device, recorded in the described first kind of Nitrite bacteria nutrient solution of sludge seeding of village's sewage disposal plant aeration tank in Tianjin, at 10 ℃, cultured continuously 5 days, take out supernatant liquor, add second kind of Nitrite bacteria nutrient solution, at 10 ℃, cultured continuously 5 days, take out supernatant liquor, add the third Nitrite bacteria nutrient solution, at 10 ℃, cultured continuously 5 days, enrichment, domestication psychrotropic bacteria;
(2) get the mud that 10mL step (1) obtains, add oneself and be equipped with in the 250mL Erlenmeyer flask of 90mL sterile purified water and 30 sterilization granulated glass spherees, vibration 20min makes bacterial cell fully be discharged in the supernatant liquor; Leave standstill 30min, get the adding of 1mL supernatant liquor and be equipped with in the test tube of 9mL sterile distilled water, obtain 10
-2Bacteria suspension, take turns doing serial dilution as stated above, accomplish 10 always
-8Till;
(3) choosing gradient is 10
-5～10
-8Bacteria suspension, get 1mL respectively and be inoculated in the Nitrite bacteria solid medium, each extent of dilution is made 3 parallel samples, the numbering; Under the condition of aerobic, preserve moisture for 10 ℃ and cultivated for 3 weeks;
(4) choosing colony is big in the Nitrite bacteria solid medium flat board from step (3), splash into the different single bacterium colony of form that griess reagent reddens, line separates again in Nitrite bacteria solid medium flat board, repeating plate streaking separates 3 times, until the homomorphosis of the bacterium colony on the flat board in microscope, single, big or small similar single bacterium colony that is, called after is inferior white 1, through be accredited as cold condition go down except sewage in the sphingolipid bacillus (Sphingobacteria sp.) of ammonia nitrogen.
Embodiment 2
First kind of Nitrite bacteria nutrient solution is made up of following component: NH
4Cl 0.0764g, sodium acetate 2g, MgSO
47H
2O0.05g, K
2HPO
40.2g, NaCl 0.12g, MnSO
4H
2O 0.01g, FeSO
40.01g adding distil water is to 1000mL.
Embodiment 3
Second kind of Nitrite bacteria nutrient solution is made up of following component: NH
4Cl 0.191g, sodium acetate 2g, MgSO
47H
2O 0.05g, K
2HPO
40.2g, NaCl 0.12g, MnSO
4H
2O 0.01g, FeSO
40.01g adding distil water is to 1000mL.
Embodiment 4
The third Nitrite bacteria nutrient solution is made up of following component: NH
4Cl 0.382g, sodium acetate 2g, MgSO
47H
2O 0.05g, K
2HPO
40.2g, NaCl 0.12g, MnSO
4H
2O 0.01g, FeSO
40.01g adding distil water is to 1000mL.
Embodiment 5
The Nitrite bacteria solid medium is made up of following component: NH
4Cl 0.0764g, sodium acetate 2g, MgSO
47H
2O 0.05g, K
2HPO
40.2g, NaCl 0.12g, MnSO
4H
2O 0.01g, FeSO
40.01g, agar 20g, adding distil water is to 1000mL.
Embodiment 6
The preparation of griess reagent
1, Sulphanilic Acid reagent (A liquid): 0.5 gram Sulphanilic Acid is dissolved in 150 milliliter 20% the dilute acetic acid solution, is stored in brown bottle, refrigerates standby.
2, alpha-naphthylamine reagent (B liquid): 0.5 gram alpha-naphthylamine is added in the dilute acetic acid solution of 20 ml distilled waters and 150 milliliter 20%, is stored in brown bottle, refrigerates standby.
3, the use liquid of griess reagent: the A liquid of getting equal proportion mixes and can use with B liquid.
Embodiment 7
Sphingolipid bacillus of the present invention has following Microbiological Characteristics:
1. morphological characteristic
Examine under a microscope, bacterium is shaft-like, long 2.6 μ m, wide 0.38 μ m; Form yellow bacterium colony at the Nitrite bacteria solid medium.
2. cultivate and learn characteristic
Under 10 ℃, when cultivating in Nitrite bacteria substratum solid medium, sphingolipid bacillus strain of the present invention has following characteristic:
The surface is more smooth, drying, and the neat in edge rule, individual small rounded.
3. physiological property
As shown in the table, aerobic condition goes down except the gram negative bacterium of ammonia nitrogen in the sewage, and the catalase positive, the methyl red test positive, hydrolyzed starch, liquefy gelatin, edwardsiella hoshinae, growth pH value are not 6～8,5～30 ℃ of growth temperatures.
| Gram | Catalase | Methyl red | The starch hydrolysis | The gelatin test | Indole test | pH | Temperature |
| G - | + | + | + | - | - | 6～8 | 5～30 |
The 16s Sequence Identification of bacterium of the present invention
16SrDNA is bacterium class prokaryotic organism very conservative sequences in very long evolutionary process, in the taxonomy of modern microorganism, playing the part of important role, as the most frequently used phyletic evolution tagged molecule of bacterial flora structural analysis, it is more efficient and convenient to carry out structural analysis of microbial community with 16S rDNA.
Carrying out Bacteria Identification with the 16s sequence is undertaken by Fig. 3.
Beef-protein medium among Fig. 3: extractum carnis 3g, peptone 10g, sodium-chlor 5g, adding distil water are to 1000mL, and pH 7.0～7.2
The LB substratum: tryptone 10g, yeast extract 5g, NaCl 10g, adding distil water is to 1000mL.
The 16s sequence that the present invention records is as follows:
ACGCTAGCGGCAGGCCTAATACATGCAAGTCGAACGATAGTGAGAAGCTTGCTTCTCACAAAAGTGG
CGCACGGGTGCGTAACGCGTATGCAACCTACCTCAATCAGGGGGATAGCCCGAAGAAATTCGGATTAA
CACCGCATAACAACACAGTACAGCATTGTACAATGTTCAAATATTTATAGGATTGAGATGGGCATGCGT
GTCATTAGCTAGTTGGCGGGGTAACGGCCCACCAAGGCGACGATGACTAGGGGATCTGAGAGGATGA
CCCCCCACACTGGTACTGAGACACGGACCAGACTCCTACGGGAGGCAGCAGTAAGGAATATTGGTCA
ATGGAGGCAACTCTGAACCAGCCATGCCGCGTGCAGGAAGACAGCCCTCTGGGTCGTAAACTGCTTT
TATTCGGGAATAAACCTTATTACGTGTAATAAGCTGAATGTACCGAAGGAATAAGGATCGGCTAACTCC
GTGCCAGCAGCCGCGGTAATACGGAGGATCCAAGCGTTATCCGGATTTATTGGGTTTAAAGGGTGCGT
AGGCGGCTTATTAAGTCAGGGGTGAAAGACGGTGGCTCAACCATCGCAGTGCCCTTGATACTGATGA
GCTTGAATGAACTAGAGGTAGGCGGAATGTGACAAGTAGCGGTGAAATGCATAGATATGTCACAGAA
CACCGATTGCGAAGGCAGCTTACTATGGTTTTATTGACGCTGAGGCACGAAAGCGTGGGGATCAAAC
AGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGAATACTCGCTGTTAGCGATATACAGTTAGCG
GCTAAGCGAAAGCGTTAAGTATTCCACCTGGGGAGTACGCCCGCAAGGGTGAAACTCAAAGGAATTG
ACGGGGGCCCGCACAAGCGGAGGAGCATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCGGG
CTTGAAAGTTAGTGAATCATTTAGAGATAGATGAGTGAGCAATCACACGAAACTAGGTGCTGCATGGC
TGTCGTCAGCTCGTGCCGTGAGGTGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATGTTTAGTTG
CCAGCACGTTAAGGTGGGGACTCTAAACAGACTGCCTGTGCAAACAGAGAGGAAGGAGGGGACGAC
GTCAAGTCATCATGGCCCTTACGTCCGGGGCTACACACGTGCTACAATGGATGGTACAGAGGGCAGC
AAGCTGGTAACAGCAAGCAAATCTCAAAAAGCCATTCACAGTTCGGATAGAGGTCTGCAACTCGACC
TCTTGAAGTTGGATTCGCTAGTAATCGCGTATCAGCAATGACGCGGTGAATACGTTCCCGGGCCTTGTA
CACACCGCCCGTCAAGCCATGGAAGTTGGGGGTACCTAAAGTATGTAACCGCAAGGAGCGTCCTAGG
G
Learn characteristic according to mentioned microorganism, inquiry uncle Jie Shi systematic bacteriology handbook, bacterial strain called after Ya Bai-1 of the present invention, classification called after sphingolipid bacillus (Aphingobacteria sp.).Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on September 22nd, 2011, deposit number is CGMCC No.5278, the preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and Institute of Microorganism, Academia Sinica, and survived.
Embodiment 8
The degradation effect of ammonia nitrogen in the Ya Bai-1 pair sewage
In the beaker of 1L, add the 4th kind of Nitrite bacteria nutrient solution of 500mL (initial ammonia nitrogen concentration is 30mg/L), add an amount of Ya Bai-1, keeping pH is 7.5, measures 72 hours degraded situations to ammonia nitrogen, the result is as shown in Figure 2.
As seen from Figure 2, the degraded through 72 hours, ammonia nitrogen concentration is 1.3mg/L in the solution, and ammonia nitrogen is removed substantially, and clearance is 95.7%, proves that the Ya Bai-1 pair ammonia nitrogen among the present invention has good removal ability.
The 4th kind of Nitrite bacteria nutrient solution formed: first kind of Nitrite bacteria nutrient solution is made up of following component: NH
4Cl0.1146g, sodium acetate 2g, MgSO
47H
2O 0.05g, K
2HPO
40.2g, NaCl 0.12g, MnSO
4H
2O 0.01g, FeSO
40.01g adding distil water is to 1000mL.
Embodiment 9
Low temperature goes down except the isolation cultivation method of the sphingolipid bacillus of ammonia nitrogen in the sewage, comprises the steps:
(1) first kind of Nitrite bacteria nutrient solution put into lab scale sbr reactor device, sludge of sewage treatment plant is inoculated in described first kind of Nitrite bacteria nutrient solution, at 8 ℃, cultured continuously 6 days, take out supernatant liquor, add second kind of Nitrite bacteria nutrient solution, at 8 ℃, cultured continuously 6 days, take out supernatant liquor, add the third Nitrite bacteria nutrient solution, at 8 ℃, cultured continuously 6 days, enrichment, domestication psychrotropic bacteria;
(2) get the mud that 10mL step (1) obtains, add oneself and be equipped with in the 250mL Erlenmeyer flask of 90mL sterile purified water and 20 sterilization granulated glass spherees, vibration 10～30min makes bacterial cell fully be discharged in the supernatant liquor; Leave standstill 20～40min, get the adding of 1mL supernatant liquor and be equipped with in the test tube of 9mL sterile distilled water, obtain 10
-2Bacteria suspension, take turns doing serial dilution as stated above, accomplish 10 always
-8Till;
(3) choosing gradient is 10
-5～10
-8Bacteria suspension, get 1mL respectively and be inoculated in the Nitrite bacteria solid medium, each extent of dilution is made 3 parallel samples, the numbering; Under the condition of aerobic, preserve moisture for 10 ℃ and cultivated for 3 weeks;
(4) choosing colony is big in the Nitrite bacteria solid medium flat board from step (3), single bacterium colony that form is different, line separates again in Nitrite bacteria solid medium flat board, repeating plate streaking separates 3 times, make the homomorphosis of bacterium colony in microscope on the flat board, single, size is similar to be single bacterium colony, through be accredited as cold condition go down except sewage in the sphingolipid bacillus (Sphingobacteria sp.) of ammonia nitrogen
Low temperature goes down except the isolation cultivation method of the sphingolipid bacillus of ammonia nitrogen in the sewage, comprises the steps:
(1) first kind of Nitrite bacteria nutrient solution put into lab scale sbr reactor device, sludge of sewage treatment plant is inoculated in described first kind of Nitrite bacteria nutrient solution, at 12 ℃, cultured continuously 4 days, take out supernatant liquor, add second kind of Nitrite bacteria nutrient solution, at 12 ℃, cultured continuously 4 days, take out supernatant liquor, add the third Nitrite bacteria nutrient solution, at 12 ℃, cultured continuously 4 days, enrichment, domestication psychrotropic bacteria;
(2) get the mud that 10mL step (1) obtains, add oneself and be equipped with in the 250mL Erlenmeyer flask of 90mL sterile purified water and 30 sterilization granulated glass spherees, vibration 10～30min makes bacterial cell fully be discharged in the supernatant liquor; Leave standstill 20～40min, get the adding of 1mL supernatant liquor and be equipped with in the test tube of 9mL sterile distilled water, obtain 10
-2Bacteria suspension, take turns doing serial dilution as stated above, accomplish 10 always
-8Till;
(3) choosing gradient is 10
-5～10
-8Bacteria suspension, get 1mL respectively and be inoculated in the Nitrite bacteria solid medium, each extent of dilution is made 3 parallel samples, the numbering; Under the condition of aerobic, preserve moisture for 10 ℃ and cultivated for 2 weeks;
(4) choosing colony is big in the Nitrite bacteria solid medium flat board from step (3), single bacterium colony that form is different, line separates again in Nitrite bacteria solid medium flat board, repeating plate streaking separates 4 times, make the homomorphosis of bacterium colony in microscope on the flat board, single, size is similar to be single bacterium colony, through be accredited as cold condition go down except sewage in the sphingolipid bacillus (Sphingobacteria sp.) of ammonia nitrogen
Experiment showed, that embodiment 9 and embodiment 10 low temperature go down the effect of good removal ammonia nitrogen is also arranged except the sphingolipid bacillus of the isolation cultivation method separation and Culture of the sphingolipid bacillus of ammonia nitrogen in the sewage, clearance is respectively 95.0% and 94.9%.
Claims (2)
1. a low temperature goes down inferior whitely 1 except the sphingolipid bacillus (Sphingobacteria sp.) of ammonia nitrogen in the sewage, and its preserving number is: CGMCC No.5278, and it has the ability of ammonia nitrogen in the low-temperature aerobic condition is gone down except sewage, and described low temperature is 8-12 ℃.
2. a kind of low temperature of claim 1 goes down except the inferior purposes of 1CGMCC No.5278 ammonia nitrogen in the low-temperature aerobic condition is gone down except sewage in vain of the sphingolipid bacillus (Sphingobacteria sp.) of ammonia nitrogen in the sewage, and described low temperature is 8-12 ℃.
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