CN113549585B - Salt-tolerant heterotrophic nitrification aerobic denitrification bacterium and application thereof - Google Patents
Salt-tolerant heterotrophic nitrification aerobic denitrification bacterium and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/02—Aerobic processes
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
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- Biodiversity & Conservation Biology (AREA)
- Hydrology & Water Resources (AREA)
- Engineering & Computer Science (AREA)
- Environmental & Geological Engineering (AREA)
- Water Supply & Treatment (AREA)
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Abstract
The invention provides a salt-tolerant heterotrophic nitrification aerobic denitrification strain, which belongs to the technical field of biology and has the preservation number of GDMCC NO.61505. The invention separates klebsiella oxytoca (Klebsiella oxytoca) from the culture wastewaterKlebsiella oxytoca) YZ-12, the strain has the salt-tolerant, heterotrophic nitrification and aerobic denitrification capabilities, can still grow and partially denitrify when the salinity is 30g/L, and has the denitrification rate of more than 97 percent for ammonia nitrogen wastewater with the concentration of less than 150mg/L and nitrate nitrogen wastewater with the concentration of less than 500 mg/L. The strain has potential application prospect in sewage treatment.
Description
Technical Field
The invention relates to a salt-tolerant heterotrophic nitrification aerobic denitrification bacterium and application thereof, belonging to the technical field of environmental microorganisms.
Background
With the increasingly prominent problem of water eutrophication, the prevention and treatment of nitrogen pollution are more and more paid attention by people. The existing sewage denitrification treatment method mainly comprises a chemical method, a biochemical method and a biophysical-physicochemical combined process, wherein the biological denitrification is a main means for sewage denitrification by the characteristics of economy, high efficiency and no residual pollution. However, the different processes of the traditional biological denitrification and denitrification require to be carried out in two reactors, so that the construction cost is high, and each step needs to be respectively added with a carbon source, acid and alkali regulation and the like.
In recent years, with the discovery of strains with heterotrophic nitrification-aerobic denitrification characteristics, the research and development of sewage denitrification technology by using heterotrophic nitrification-aerobic denitrification bacteria is increasingly a research hotspot. Compared with the traditional nitrifying microorganisms, the heterotrophic nitrification aerobic denitrifying bacteria have higher cell growth rate, realize synchronous completion of nitrification and denitrification reactions in the same reactor, and simultaneously convert ammonia nitrogen, nitrate nitrogen and nitrite nitrogen into nitrogen-containing gas. In addition, acid and alkali respectively generated in the nitrification process and the denitrification process can be mutually neutralized, so that the acid-alkali balance of the water body is relatively maintained, and the pH adjustment cost is reduced. Some special heterotrophic nitrification aerobic denitrification bacteria even have the characteristics of high ammonia nitrogen tolerance, high salinity tolerance, low temperature tolerance and low C/N tolerance. These advantages make heterotrophic nitrification aerobic denitrification bacteria become a research hotspot for sewage denitrification treatment.
Disclosure of Invention
The invention provides a salt-tolerant heterotrophic nitrification aerobic denitrification strain which can be applied to the field of sewage treatment.
Klebsiella oxytoca (Klebsiella oxytoca, etc.) provided by the present inventionKlebsiella oxytoca) YZ-12, deposited at 21.7.2021 in Guangdong province, gmbH, with accession number GDMCC NO.61505.
The screened Klebsiella oxytoca YZ-12 is applied to sewage treatment.
The invention also provides a method for denitrifying sewage by applying the Klebsiella oxytoca YZ-12, which comprises the following steps:
(1) Activating acid-producing Klebsiella bacteria YZ-12; specifically, klebsiella oxytoca YZ-12 can be inoculated into a solid culture medium and cultured for 24 hours at the temperature of 28-35 ℃ to prepare an activated strain; the solid culture medium is prepared by adding 15-20 g/L agar powder into LB culture medium.
(2) Preparing a seed solution; specifically, the activated strain prepared in the step (1) is inoculated in a seed culture medium and cultured for 12-16 h at the temperature of 28-35 ℃ and the speed of 120-200 r/min, and then seed liquid is obtained; the seed medium may be: 1g/L tryptone, 0.5g/L yeast extract, 0.1g/L potassium nitrate and pH 7.0-7.5.
(3) Carrying out denitrification treatment on the sewage; specifically, inoculating the seed solution obtained in the step (2) into sewage to be treated according to the volume ratio of 5%, adding sucrose as a carbon source, adjusting the C/N to be 10-20 and the pH to be 6-9, culturing for 24-48 h at the temperature of 28-35 ℃ and under the condition of 120-200 r/min, and taking a water sample to detect the denitrification effect of the strain.
The Klebsiella oxytoca YZ-12 has certain high salt tolerance and is contained in 30 g/mlThe strain number can reach 10 after the culture is carried out for 48 hours in the L sodium chloride denitrification culture medium 8 cell/mL。
The invention has the beneficial effects that:
the Klebsiella oxytoca YZ-12 provided by the invention has the characteristics of being very suitable for sewage denitrification treatment. The living bacteria have salt tolerance, and have good nitrification capacity when the salinity is 10g/L, and good denitrification capacity when the salinity is less than or equal to 30 g/L; the strain also has a good acid-base adaptation range, and has a good denitrification effect within the pH range of 6-9; the most suitable carbon source is sucrose; the optimal C/N range for denitrification is 10-20.
Drawings
FIG. 1 bacterial strain YZ-12 plate colony morphology.
FIG. 2 PCR gel electrophoresis image of strain YZ-12.
FIG. 3 phylogenetic tree of strain YZ-12.
FIG. 4 the effect of different carbon sources on the denitrification effect of strain YZ-12.
FIG. 5 Effect of different initial nitrogen solubility on the denitrification effect of strain YZ-12.
FIG. 6 shows the effect of different carbon nitrogen ratios on the denitrification effect of the strain YZ-12.
FIG. 7 is a graph showing the effect of different pH values on the denitrification effect of the strain YZ-12.
FIG. 8 the effect of different salinity on the denitrification effect of strain YZ-12.
FIG. 9 shows the denitrification effect of the strain YZ-12 on the culture wastewater.
Detailed Description
The following examples further illustrate the invention. The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a process are given, but the scope of the present invention is not limited to the following embodiments.
Example 1
Screening, identifying and storing Klebsiella oxytoca YZ-12
(1) Screening of Klebsiella oxytoca YZ-12
Taking a water sample of an aerobic pool of a farm sewage treatment station, adding the water sample into a sterile primary screening culture medium according to the inoculation amount of 10%, carrying out constant-temperature culture on a 120rpm shaking table at 30 ℃ for 24 hours,passage was continued 3 times. Diluting the culture solution obtained by continuous culture for 3 times with sterile water 10 times -5 、10 -6 、10 -7 And coating the single colony on a primary screening solid culture medium, culturing at the constant temperature of 30 ℃ for 1-2 days, selecting the blue single colony on the colony area when the single colony grows on the flat plate, streaking on the primary screening solid culture medium, repeatedly picking the single colony to streak again when the single colony grows on the flat plate, finally streaking on an LB solid culture medium test tube inclined plane, and storing in a refrigerator at 4 ℃.
The formula of the primary screening culture medium is as follows: c 4 H 4 Na 2 O 4 ·6H 2 O 8.5g/L，KNO 3 1g/L，MgSO 4 ·7H 2 O 1g/L，KH 2 PO 4 1g/L，CaCl 2 ·2H 2 O 0.2g/L，FeSO 4 ·7H 2 O0.05 g/L,1% bromothymol blue 1mL/L, pH 7.0.
The formula of the primary screening solid culture medium is that agar is added by 20g/L on the basis of the formula of the primary screening culture medium.
(2) Identification of Klebsiella oxytoca YZ-12
As shown in the figure I, the bacterial colony of the strain YZ-12 on an LB medium plate is round, white, smooth in surface, viscous, elastic and capable of being pulled into a filamentous shape. The thallus is rod-shaped under microscope.
The selected strains were subjected to 16S rDNA sequencing. After PCR amplification, the obtained 16S rDNA sequence is compared on NCBI and then is subjected to phylogenetic tree construction (see figure III), and the strain is determined to be Klebsiella oxytoca YZ-12.
(3) Preservation of Klebsiella oxytoca YZ-12
Selecting and inoculating Klebsiella oxytoca YZ-12 strain in LB culture medium, culturing at 30 deg.C and 120rpm for 12-24 hr, transferring 0.6mL culture solution into a 40% glycerol storage tube containing 0.6mL, and freezing at-20 deg.C. The strain is preserved in Guangdong province microorganism culture collection center at 2021, 7 months and 21 days, and the preservation number is GDMCC NO.61505.
Example 2
Denitrification effect of Klebsiella oxytoca YZ-12 under different conditions
(1) Detection of influence of carbon source on denitrification effect of Klebsiella oxytoca YZ-12
Preparing a seed solution: the Klebsiella oxytoca YZ-12 stored in the refrigerator at the temperature of 4 ℃ in the example 1 is taken and inoculated into a 250mL conical flask filled with 50mL of seed culture medium, and the culture is carried out for 12 to 16 hours under the conditions of 28 to 35 ℃ and 120 to 200r/min, thus obtaining the seed liquid.
And (3) denitrification effect detection: preparing ammonia nitrogen culture medium and nitrate nitrogen culture medium by using glucose, sucrose, sodium acetate, methanol and sodium succinate as unique carbon sources, and preparing NH 4 + -N concentration of 350mg/L, NO 3 - the-N concentration is 415mg/L, the seed liquid is added according to the inoculation amount of 5 percent, the mixture is cultured for 72 hours under the conditions of 28-35 ℃ and 120-200 r/min, sampling is carried out every 24 hours, centrifugation is carried out at 8000rpm, supernatant is taken, and NH is detected by a nano-reagent spectrophotometry 4 + -N concentration, detection of NO by ion chromatography 3 - -N concentration. Wherein the sucrose is used as a carbon source and NH is contained in an ammonia nitrogen culture medium 4 + The removal rate of-N is 42.86 percent, and NO is contained in the nitrate nitrogen culture medium by taking cane sugar as a carbon source 3 - The removal rate of-N is 99.84%, and neither the ammonia nitrogen culture medium nor the nitrate nitrogen culture medium has NO 2 - -N accumulation.
(2) Detection of influence of nitrogen source concentration on denitrification effect of Klebsiella oxytoca YZ-12
Preparation of NH 4 + N concentration gradient of 50, 100, 150, 200, 250mg/L, NO 3 - Adding an ammonia nitrogen culture medium and a nitrate nitrogen culture medium of sucrose with the N concentration of 400, 450, 500, 550, 600mg/L and 9.8g/L into the seed solution according to the inoculation amount of 5 percent, culturing for 72 hours under the conditions of 28-35 ℃ and 120-200 r/min, sampling every 24 hours, centrifuging at 8000rpm, taking supernatant, and detecting NH by using a Nashin reagent spectrophotometry 4 + -N concentration, detection of NO by ion chromatography 3 - -N concentration. Wherein NH 4 + NH at a N concentration of 50, 100mg/L 4 + The removal rate of-N is more than 99 percent, NO 3 - NO at N concentration of 400, 450mg/L 3 - The removal rate of-N is 99%, and NO NO is generated 2 - -N accumulation.
(3) Detection of influence of C/N on denitrification effect of Klebsiella oxytoca YZ-12
Fixation of NH 4 + N concentration of 100mg/L, NO 3 - The concentration of N is 415mg/L, and the C/N is adjusted to be 2.5, 5, 10, 15 and 20. Adding the seed solution according to the inoculation amount of 5 percent, culturing for 72 hours under the conditions of 28-35 ℃ and 120-200 r/min, sampling every 24 hours, centrifuging at 8000rpm, taking supernatant, and detecting NH by using a nano-grade reagent spectrophotometry 4 + -N concentration, detection of NO by ion chromatography 3 - -N concentration. Wherein when C/N is 10, 15, 20, NH 4 + -N and NO 3 - The removal rate of-N is more than 99 percent, and NO NO is generated 2 - -N accumulation.
(4) Detection of influence of pH on effect of Klebsiella oxytoca YZ-12 nitrogen
Configuration of NH 4 + N concentration of 100mg/L, NO 3 - Ammoniacal nitrogen medium, nitrate nitrogen medium with N concentration 415mg/L and C/N10, adjusted to pH =5, pH =6, pH =7, pH =8, pH =9 with 1mol/L hydrochloric acid solution and 1mol/L sodium hydroxide solution, respectively. Adding the seed solution according to the inoculation amount of 5 percent, culturing for 72 hours under the conditions of 28-35 ℃ and 120-200 r/min, sampling every 24 hours, centrifuging at 8000rpm, taking supernatant, and detecting NH by using a nano-grade reagent spectrophotometry 4 + -N concentration, detection of NO by ion chromatography 3 - -N concentration. Wherein NH when the pH is 7, 8 or 9 4 + -N and NO 3 - The removal rate of-N is more than 99 percent, and NO NO is generated 2 - -N accumulation.
(5) Detection of influence of salinity on denitrification effect of Klebsiella oxytoca YZ-12
Configuration of NH 4 + N concentration of 100mg/L, NO 3 - An ammonia nitrogen culture medium and a nitrate nitrogen culture medium with the N concentration of 415mg/L and the C/N of 10 are added with NaCl with the mass ratio of 1%, 2%, 3%, 4% and 5%, respectively. Adding the seed solution according to the inoculation amount of 5 percent, culturing for 72 hours under the conditions of 28-35 ℃ and 120-200 r/min, sampling every 24 hours, centrifuging at 8000rpm, taking supernatant, and detecting NH by using a nano-grade reagent spectrophotometry 4 + -N concentration, detection of NO by ion chromatography 3 - -N concentration. Wherein the salinity is 1% NH 4 + -N and NO 3 - The N removal rate is more than 99 percent; NH when salinity is 2% 4 + the-N removal rate was 66.7%, NO 3 - -N removal 99.9%; NH when salinity is 3% 4 + -N removal of 23.0%, NO 3 - The removal rate of-N is 99.7%, and NO NO is generated 2 - -N accumulation.
The formula of the culture medium comprises:
seed culture medium: tryptone 1g/L, yeast extract 0.5g/L, KNO 3 0.1g/L, pH 7.0.
An ammonia nitrogen culture medium: c 4 H 4 Na 2 O 4 ·6H 2 O 11g/L，Na 2 HPO 4 ·12H 2 O 6.7g/L，KH 2 PO 4 1g/L，NH 4 Cl 1.5g/L，MgSO 4 ·7H 2 O0.1 g/L, trace element solution 2mL, pH 7.0-7.3.
Nitrate nitrogen culture medium: sucrose 5g/L, na 2 HPO 4 ·12H 2 O 7.9g/L，KH 2 PO 4 1.5g/L，MgSO 4 ·7H 2 O 0.1g/L，KNO 3 3g/L, 2mL of trace element solution and 7.0-7.3 of pH value.
Trace elements: EDTA 50g/L, caCl 2 ·2H 2 O 7.28g/L，FeSO 4 ·7H 2 O 5g/L，ZnSO 4 ·7H 2 O 3.92g/L，MnCl 2 ·4H 2 O 2.06g/L，CoCl 2 ·6H 2 O 1.61g/L，CuSO 4 ·5H 2 O 1.57g/L，(NH 4 ) 6 Mo 7 O 24 ·4H 2 O1.1 g/L, pH 6.0.
Example 3
Klebsiella oxytoca YZ-12 denitrification treatment effect on aquaculture wastewater
Adjusting the C/N of the culture wastewater to 10 by using sucrose, adding the seed solution of the example 2 according to the inoculation amount of 5 percent, culturing for 72 hours under the conditions of 28-35 ℃ and 120-200 r/min, sampling every 24 hours, centrifuging at 8000rpm, taking supernatant, and detecting NH by using a Nashin reagent spectrophotometry 4 + -N concentration, detection of NO by ion chromatography 3 - -N concentration. Fruiting aquaculture wastewater NH 4 + The removal rate of-N was 95.15%, NO 3 - the-N removal rate was 99.76%.
The water quality condition of the aquaculture wastewater is as follows: the pH value is 7.8, the COD value is 2031mg/L, the ammonia nitrogen is 362mg/L, the nitrate nitrogen is 23.8mg/L, and the nitrite nitrogen is 0.167mg/L.
The foregoing is a further detailed description of the invention in connection with specific preferred embodiments and it is not intended to limit the invention to the specific embodiments described. For those skilled in the art to which the invention pertains, numerous simple deductions or substitutions may be made without departing from the spirit of the invention, which shall be deemed to belong to the scope of the invention.
Sequence listing
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ggtgcaagcg ttaatcggaa ttactgggcg taaagcgcac gcaggcggtc tgtcaagtcg 540
gatgtgaaat ccccgggctc aacctgggaa ctgcattcga aactggcagg ctggagtctt 600
gtagaggggg gtagaattcc aggtgtagcg gtgaaatgcg tagagatctg gaggaatacc 660
ggtggcgaag gcggccccct ggacaaagac tgacgctcag gtgcgaaagc gtggggagca 720
aacaggatta gataccctgg tagtccacgc tgtaaacgat gtcgacttgg aggttgttcc 780
cttgaggagt ggcttccgga gctaacgcgt taagtcgacc gcctggggag tacggccgca 840
aggttaaaac tcaaatgaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat 900
tcgatgcaac gcgaagaacc ttacctactc ttgacatcca gagaacttag cagagatgct 960
ttggtgcctt cgggaactct gagacaggtg ctgcatggct gtcgtcagct cgtgttgtga 1020
aatgttgggt taagtcccgc aacgagcgca acccttatcc tttgttgcca gcgattcggt 1080
cgggaactca aaggagactg ccagtgataa actggaggaa ggtggggatg acgtcaagtc 1140
atcatggccc ttacgagtag ggctacacac gtgctacaat ggcatataca aagagaagcg 1200
acctcgcgag agcaagcgga cctcataaag tatgtcgtag tccggattgg agtctgcaac 1260
tcgactccat gaagtcggaa tcgctagtaa tcgtggatca gaatgccacg gtgaatacgt 1320
tcccgggcct tgtacacacc gcccgtcaca ccatgggagt gggttgcaaa agaagtaggt 1380
agcttaacct tcgggagggc gctacc 1406
Claims (2)
1. A strain of salt-tolerant heterotrophic nitrification aerobic denitrification bacteria is characterized in that: the strain is Klebsiella oxytoca (A), (B), (C) and (C)Klebsiella oxytoca) YZ-12, the strain is preserved in Guangdong province microorganism strain preservation center, and the strain preservation number is GDMCC NO.61505.
2. The application of the salt-tolerant heterotrophic nitrification-aerobic denitrification bacterium as claimed in claim 1 in sewage treatment as a sewage treatment microbial inoculum.
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| WO2011054401A1 (en) * | 2009-11-09 | 2011-05-12 | Akaeno Sas | Bacterial composition for treating blood-containing fatty effluents |
| CN103013867A (en) * | 2012-12-04 | 2013-04-03 | 南京农业大学 | Acid-producing klebsiella pneumoniae DF-1 and application thereof in removing nitrous nitrogen in water body |
| CN106834158A (en) * | 2016-08-12 | 2017-06-13 | 轻工业环境保护研究所 | A kind of microbial bacterial agent for processing leather waste water and preparation method thereof |
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| WO2014089025A1 (en) * | 2012-12-04 | 2014-06-12 | Genomatica, Inc. | Increased yields of biosynthesized products |
| CN108102943B (en) * | 2017-10-11 | 2021-04-23 | 四川大学 | Denitrifying microorganism and application thereof |
| CN112063550A (en) * | 2020-08-24 | 2020-12-11 | 郑州轻工业大学 | For reducing Fe in complex stateⅢAnaerobic strain, culture method and application thereof |
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| WO2011054401A1 (en) * | 2009-11-09 | 2011-05-12 | Akaeno Sas | Bacterial composition for treating blood-containing fatty effluents |
| CN103013867A (en) * | 2012-12-04 | 2013-04-03 | 南京农业大学 | Acid-producing klebsiella pneumoniae DF-1 and application thereof in removing nitrous nitrogen in water body |
| CN106834158A (en) * | 2016-08-12 | 2017-06-13 | 轻工业环境保护研究所 | A kind of microbial bacterial agent for processing leather waste water and preparation method thereof |
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