CN109337832B - High-ammonia-nitrogen-resistant heterotrophic nitrification-aerobic denitrification ochrobactrum and application thereof - Google Patents
High-ammonia-nitrogen-resistant heterotrophic nitrification-aerobic denitrification ochrobactrum and application thereof Download PDFInfo
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Abstract
The invention relates to high ammonia nitrogen tolerant heterotrophic nitrification-aerobic denitrification ochrobactrum TAC-2 (Ochrobactrum sp.TAC-2), and the preservation number is CCTCC NO: M2018028. The bacteria can realize the process of synchronous nitrification and denitrification in an aerobic environment under the conditions of high ammonia nitrogen and high salinity, effectively remove total nitrogen in high ammonia nitrogen wastewater and high salinity wastewater, are particularly suitable for removing ammonia nitrogen in high ammonia nitrogen wastewater, and have the significance of wide popularization.
Description
Technical Field
The invention belongs to the technical field of environmental microorganisms, and particularly relates to high ammonia nitrogen resistant heterotrophic nitrification-aerobic denitrification ochrobactrum and application thereof.
Background
In recent years, the discharge amount of high ammonia nitrogen wastewater (ammonia nitrogen concentration is more than 200 mg/L) is increasing. The high ammonia nitrogen wastewater is improperly treated and directly discharged into the water body, so that the phenomena of water body eutrophication and the like become serious day by day, and the water body quality is deteriorated. In order to control the ammonia nitrogen pollution of water bodies, new strict pollutant control standards are issued in China, wherein the primary standard of ammonia nitrogen is 5.0 mg/L. The new standard provides new requirements for the effective control of ammonia nitrogen, in particular to the effective control of ammonia nitrogen in high ammonia nitrogen wastewater.
Due to the problems of high cost and secondary pollution, the physical and chemical methods for removing ammonia nitrogen in high ammonia nitrogen wastewater (such as a stripping method, an ammonia distillation tower distillation method, a breakpoint chlorination method, a FENTON oxidation method and the like) are only applied to special chemical industries. Biological denitrification is regarded as the most promising water body denitrification method at present due to the advantages of no pollution, economy, safety and the like.
The traditional biological denitrification technology mainly comprises an A/O (anaerobic-aerobic) process, a shortcut nitrification-denitrification process, a nitrification and anaerobic ammonia oxidation process and the like. The biological denitrification technologies all utilize the characteristics of autotrophic nitrifying bacteria to achieve the aim of removing ammonia nitrogen. However, a large amount of free ammonia contained in the high-concentration ammonia-nitrogen wastewater has a strong inhibiting effect on autotrophic nitrifying bacteria, so that the ammonia oxidation process of the autotrophic nitrifying bacteria is inhibited, the ammonia-nitrogen treatment effect is influenced, and the treatment effect of the biological methods is poor when the high-ammonia-nitrogen wastewater is treated. In addition, the biological denitrification processes do not get rid of the two-stage biological denitrification limitation of aerobic-anaerobic combination, and a large amount of space is needed for separating the storage tanks of the aerobic environment and the anaerobic environment, so that the construction cost of denitrification equipment is higher.
In recent years, researchers find special heterotrophic nitrification-aerobic denitrification bacteria which have the characteristics of synchronous nitrification and denitrification, can synchronously remove ammonia nitrogen and total nitrogen in one reactor under the completely aerobic condition, and solve the contradiction between the nitrification process and the denitrification process. With the continuous and deep research, the bacteria have the biochemical characteristics of salt resistance, low temperature resistance, high ammonia nitrogen resistance, poor nutrition environment resistance and the like.
The ochrobactrum is a flora which is obligate and aerobic, has strict respiratory metabolism and can utilize various amino acids, organic acids and carbohydrates as carbon sources, and the heterotrophic nitrification-aerobic denitrification function of the ochrobactrum is not recorded in documents at present.
Disclosure of Invention
The invention aims to provide the canebright bacillus of high ammonia nitrogen resistant heterotrophic nitrification-aerobic denitrification, which can realize the process of synchronous nitrification and denitrification in an aerobic environment under the conditions of high ammonia nitrogen and high salinity, effectively remove the total nitrogen in high ammonia nitrogen wastewater and high salinity wastewater, is particularly suitable for removing the ammonia nitrogen in the high ammonia nitrogen wastewater, and has the significance of wide popularization.
The Total Nitrogen (TN) in the invention refers to ammonia Nitrogen (NH)4 +-N\ NH3-N), nitrate Nitrogen (NO)3 -) Nitrite Nitrogen (NO)2 -) The sum of (a) and (b).
The technical scheme of the invention is as follows:
the high ammonia nitrogen resistant heterotrophic nitrification-aerobic denitrification ochrobactrum has the preservation number of CCTCC NO: M2018028 (preserved in China center for type culture Collection in 2018, 1, 12 and the address: Wuhan university in Wuhan, China), and is classified and named as:Ochrobacterumsp, TAC-2, the 16S rDNA sequence of which is shown in SEQ ID NO 1.
The invention relates to ochrobactrum anthropi resistant to high ammonia nitrogen heterotrophic nitrification-aerobic denitrification, belonging to the genus ochrobactrum. The colony diameter is about 1 mm, and the colony is semitransparent, protruding, neat in edge, glossy in surface, white and transparent in color, and gram-negative. The optimal growth temperature is 25-40 ℃, and the pH is 6.5-7.5.
The ochrobactrum anthropi TAC-2 has strong tolerance to high ammonia nitrogen and high salinity, takes organic matters as an electron acceptor, NH4 +As electron donor, adding NH4 +By oxidation to NO2 -Or NO3 -(ii) a Meanwhile, in an aerobic environment, organic matters are taken as electron donors, NO2 -Or NO3 -As electron acceptor, NO2 -Or NO3 -Reducing to nitrogen. The ochrobactrum TAC-2 can be applied to efficient denitrification treatment of high-ammonia-nitrogen wastewater and high-salinity wastewater.
The ochrobactrum anthropi of the invention is named as ochrobactrum anthropi in the applicationOchrobacterumsp, TAC-2, which is separated from biogas slurry in a pig farm, and the sequencing of the strain DNA is completed by the Shinto Dalibao biological company, and the 16S rDNA sequence of the strain DNA is shown as SEQ ID NO. 1. A series of physiological and biochemical tests and process optimization tests carried out on the strain show that the ochrobactrum provided by the inventionOchrobacterumsp, TAC-2, can completely remove the total nitrogen in the high ammonia nitrogen wastewater and the high salinity wastewater, and has the following main advantages:
(1) ochrobactrum anthropiOchrobacterumThe sp, TAC-2 can grow by taking various amino acids, organic acids and carbohydrates as carbon sources and ammonia nitrogen as a unique nitrogen source, and overcomes the defects of slow growth, long generation period, low biomass concentration, poor environmental adaptability and the like of the traditional nitrobacteria.
(2) Ochrobactrum anthropiOchrobacterumsp, TAC-2 can remove total Nitrogen (NH) in a completely aerobic environment4 +) Nitrate Nitrogen (NO)3 -) Nitrite Nitrogen (NO)2 -) And the two-stage biological denitrification limitation of the traditional biological denitrification process combining aerobic and anaerobic processes is solved.
(3) Ochrobactrum anthropiOchrobacterumThe sp, TAC-2 has high ammonia nitrogen and high salinity tolerance, and can efficiently remove the total nitrogen in the high ammonia nitrogen wastewater and the high salinity wastewater.
(4) Ochrobactrum anthropiOchrobacterumThe culture medium required by the sp, TAC-2 expanding culture has simple components and low cost. Is particularly suitable for biological denitrification of high ammonia nitrogen wastewater, and can be widely popularized and applied.
The invention solves the problems of poor treatment effect, complex process, high cost and the like in the existing biological denitrification technology of high ammonia nitrogen wastewater.
In order to make the aforementioned and other objects, features and advantages of the present invention comprehensible, preferred embodiments accompanied with figures are described in detail below.
Drawings
FIG. 1 shows Ochrobactrum anthropiOchrobacterumsp, TAC-2, the ammonia nitrogen and total nitrogen removal curve when the ammonia nitrogen concentration is 400 mg/L;
FIG. 2 shows Ochrobactrum anthropiOchrobacterumsp, TAC-2 and ammonia nitrogen removal curves with different concentrations.
FIG. 3 shows Ochrobactrum anthropiOchrobacterumsp, TAC-2, ammonia nitrogen removal curve under different salinity.
Detailed Description
The present invention is further described below with reference to examples, but the embodiments of the present invention are not limited thereto.
1. Experimental Material
The culture medium for the ochrobactrum comprises the following components: (NH)4)2SO4 2 g/L，Na3C6H5O710.64 g/L, 50 mL/L Vickers salt solution, wherein Vickers salt solution (g/L): k2HPO4 5.0，MgSO4 7H2O 2.5，NaCl 2.5，FeSO4 7H2O 0.05，MnSO4 4H2And O is 0.05. Adjusting the pH value of the culture medium of the ochrobactrum to 7.0.
Besides sodium acetate, the carbon source can also directly utilize glucose, sucrose, sodium citrate and other common carbon sources.
The rest reagents are commercial analytical pure products.
Biogas slurry samples from pig farm were obtained from Chongqing Wood elk pig farm (Jiang Jiasi village in Banan, Chongqing City).
2. Strain enrichment and optimization experiment
1) Enrichment culture: inoculating 2 mL of biogas slurry in pig farm into 250 mL triangular flask containing 100 mL of sterilized broth culture medium, shaking thoroughly, culturing at 30 deg.C under 150 r.min-1, and storing in refrigerator at 4 deg.C after 2 d. Then, the enriched microorganisms were inoculated in a 250 mL Erlenmeyer flask containing 100 mL of sterilized culture medium for Xanthium sibiricum at an inoculum size of 2%. Subculturing under the same conditions, and enriching and subculturing for 5 times.
2) And (3) pure bacteria separation: diluting the enriched bacteria liquid with cooled sterile distilled water by 10 times to obtain 10-fold dilution-1、10-2、10-3、10-4、10-5、10-6、10-7The diluent (2). Culture of the culture medium of the ochrobactrum anthropi is carried out by adopting a pouring method. The plate was placed in a biochemical incubator and incubated at 30 ℃ for 2 days. And (4) carrying out multiple passages on the strains with good growth vigor, and purifying.
3) Optimization experiment: preparing bacterial suspension with a certain concentration by using pure strains, inoculating the bacterial liquid into a 250 mL conical flask filled with 100 mL of sterilized ochrobactrum culture medium according to 2 percent, fully shaking up, and culturing under the conditions of 30 ℃ and 150 r.min < -1 >. Setting ammonia nitrogen concentrations to be 100 mg/L, 200 mg/L, 400 mg/L, 600 mg/L, 800 mg/L and 1000 mg/L respectively; the salinity is respectively set as: 1%, 2%, 3%, 4%, 5%; only a single factor is changed, and the concentration of the bacteria liquid and the concentration of ammonia nitrogen are detected at intervals.
3. Bacterial species identification experiment
The target fragment was amplified by PCR using QIAquick Genomic DNA Buffer Set. Mu.l of the DNA fragment was subjected to 3% agarose gel electrophoresis, and the desired fragment was recovered by using a cut gel to perform DNA sequencing. Sequencing of DNA was performed by Competition Dalianbao Biometrics. DNA sequencing was carried out using Seq Forward, Seq Reverse, and Seq Internal as primers. The 16S rRNA amplification adopts broad-spectrum primers F27 (-AGAGTTTGATCATGGCTCAG) with the sequence shown in SEQ ID NO. 2 and R1492 (-TACGGTTACCTTGTTACGACTT) with the sequence shown in SEQ ID NO. 3.
4. Detection method
And detecting the OD value of the bacterial liquid by using a UV2000 spectrophotometer, wherein the wavelength is 600 nm. Reference is made to "Water and wastewater monitoring and analysis method" (fourth edition, published by Chinese environmental science, 2002) for methods for monitoring and analyzing various pollutants.
Example 1 Ochrobactrum anthropiOchrobacterumAmmonia of sp, TAC-2 at ammonia nitrogen concentration of 400 mg/LNitrogen and Total Nitrogen removal Capacity determination experiment
Preparing a ochrobactrum culture medium with ammonia nitrogen concentration of 400 mg/L, taking 100 mL of the ochrobactrum culture medium in a 250 mL conical flask, sterilizing the ochrobactrum culture medium with high-temperature steam at 121 ℃ for 30 min, cooling, and adding 2 mL of ochrobactrum liquid (OD) into the flask by using a micropipettor600 nm 1-2), sealing with sealing film, placing in a shaking table at 30 deg.C and 150 r min-1Culturing under the condition, and measuring OD of bacterial liquid every 24 h600 nmAnd determining the growth condition of the bacteria, simultaneously determining the contents of ammonia nitrogen and total nitrogen in the culture medium, and determining the removal effect of the ammonia nitrogen and the total nitrogen. As can be seen from FIG. 1, Ochrobactrum anthropiOchrobacterum400 mg/L ammonia nitrogen is completely removed by sp, TAC-2, and the total nitrogen removal rate is more than 90%.
Example 2 Ochrobactrum anthropiOchrobacterumExperiment of sp, TAC-2 effect of removing ammonia nitrogen with different concentrations
Respectively preparing culture medium of Xanthium sibiricum with ammonia nitrogen concentration of 100 mg/L, 200 mg/L, 400 mg/L, 600 mg/L, 800 mg/L and 1000 mg/L, respectively taking 100 mL of Xanthium sibiricum with different ammonia nitrogen concentrations to culture in 250 mL conical flask, sterilizing with high temperature steam at 121 deg.C for 30 min, cooling, adding 2 mL of Xanthium sibiricum bacterial liquid (OD) into the flask by micropipettor600 nm 1-2), sealing with sealing film, placing in a shaking table at 30 deg.C and 150 r min-1Culturing under the condition, and measuring OD of bacterial liquid every 24 h600 nmAnd determining the growth condition of the bacteria, measuring the content of ammonia nitrogen in the culture medium and determining the removal effect of the ammonia nitrogen. As can be seen from FIG. 2, when the ammonia nitrogen concentration is less than 600 mg/L, the ammonia nitrogen removal rate is 100%, and the ochrobactrum anthropiOchrobacterumThe maximum ammonia nitrogen tolerance concentration of sp, TAC-2 can reach 800 mg/L, and the ammonia nitrogen removal rate is 34.2%, which is mainly because the high-concentration ammonia nitrogen culture medium contains a large amount of free ammonia to inhibit the activity of the strain.
Example 3 Ochrobactrum anthropiOchrobacterumExperiment for high ammonia nitrogen removal effect of sp, TAC-2 under different salinity conditions
Respectively preparing the culture media of the ochrobactrum anthropi with the salinity of 1%, 2%, 3%, 4% and 5%, wherein the ammonia nitrogen concentration of each culture medium of the ochrobactrum anthropi is equal to that of400 mg/L, respectively taking 100 mL of Ochrobactrum anthropi culture medium with different salinity, sterilizing with high temperature steam at 121 deg.C for 30 min, cooling, and adding 2 mL of Ochrobactrum anthropi bacterial liquid (OD) into the bottle with micropipettor600 nm 1-2), sealing with sealing film, placing in a shaking table at 30 deg.C and 150 r min-1Culturing under the condition, and measuring OD of bacterial liquid every 24 h600 nmAnd determining the growth condition of the bacteria, measuring the content of ammonia nitrogen in the culture medium and determining the removal effect of the ammonia nitrogen. As can be seen from FIG. 3, when the salinity is less than 2%, the ammonia nitrogen removal rate is 85.7%, and the Xanthium sibiricum is usedOchrobacterumThe sp, TAC-2 maximum salinity tolerance concentration can reach 2%, and the ammonia nitrogen removal rate is 33.8%, because the microbial cell walls are separated due to the high osmotic pressure of the high salinity culture medium, so that the growth of the microbial cell walls is hindered and even stopped.
The ochrobactrum anthropi TAC-2 screened from the biogas slurry in the pig farm has strong tolerance to high ammonia nitrogen and high salinity, can metabolize by using an organic carbon source as a unique carbon source and ammonia nitrogen as a unique nitrogen source, completely removes the high ammonia nitrogen through heterotrophic nitrification-aerobic denitrification, and simultaneously realizes total nitrogen removal. The bacterial strain can also take nitrate nitrogen and nitrite nitrogen as unique nitrogen sources, and efficiently remove the nitrate nitrogen and the nitrite nitrogen through aerobic denitrification. The strain is applied to the treatment of high ammonia nitrogen wastewater and high salinity wastewater, can realize the synchronous removal of ammonia nitrogen and total nitrogen under a single aerobic condition, is beneficial to solving the problem of biological denitrification under the high ammonia nitrogen condition, and has wide application prospect.
The above list is only three specific embodiments of the present invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Sequence listing
<110> Chongqing university of science and technology
<120> ochrobactrum anthropi resistant to high ammonia nitrogen heterotrophic nitrification-aerobic denitrification and application thereof
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<170> SIPOSequenceListing 1.0
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gtggggaata ttgcacaatg ggggaaaccc tgatgcagca acgccgcgtg agtgatgacg 60
gtcttcggat tgtaaagctc tgtctttggg gacgataatg acggtaccca aggaggaagc 120
cacggctaac tacgtgccag cagccgcggt aatacgtagg tggcgagcgt tgtccggatt 180
tactgggcgt aaagggagcg taggcggatt cttaagtggg atgtgaaata cctgggctta 240
acctgggtgc tgcattccaa actgggaatc tagagtgcag gaggggagag tggaattcct 300
agtgtagcgg tgaaatgcgt agagattagg aagaacacca gtggcgaagg cgactctctg 360
gactgtaact gacgctgagg ctcgaaagcg tggggagcaa acaggattag aaacccctgt 420
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tacggttacc ttgttacgac tt 22
Claims (4)
1. Caneberry albiflora (caneberry) resistant to high-ammonia-nitrogen heterotrophic nitrification-aerobic denitrificationOchrobacterum sp) TAC-2 with the preservation number of CCTCC NO: M2018028.
2. The ochrobactrum of claim 1 (f), (f)Ochrobacterum sp) Application of TAC-2 in removing total nitrogen in high ammonia nitrogen wastewater, wherein the concentration of ammonia nitrogen in the high ammonia nitrogen wastewater is less than or equal to 800 mg/L.
3. The ochrobactrum of claim 1 (f), (f)Ochrobacterum sp) Application of TAC-2 in removing total nitrogen in high-salinity wastewater and ochrobactrum anthropi thereofOchrobacterumThe maximum salinity tolerance concentration of the sp, TAC-2 is less than or equal to 2 percent.
4. The ochrobactrum of claim 1 (f), (f)Ochrobacterum sp) Application of TAC-2 in preparation of a preparation for removing ammonia nitrogen and/or total nitrogen in wastewater.
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| CN111233148A (en) * | 2020-01-19 | 2020-06-05 | 重庆理工大学 | Efficient biomembrane synchronous nitrification and denitrification low-carbon sewage denitrification process |
| CN113388553B (en) * | 2021-07-28 | 2023-05-16 | 重庆理工大学 | Ammonia nitrogen-resistant composite microbial inoculant, application and preparation method thereof |
| CN113416681B (en) * | 2021-07-28 | 2023-05-19 | 重庆理享生态修复研究院有限公司 | Heterotrophic nitrification-aerobic denitrification bacteria resistant to low carbon and high nitrogen and application thereof |
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