CN114214260B - Halomonas LYX-3 separated from soil of smelting plant and application thereof - Google Patents
Halomonas LYX-3 separated from soil of smelting plant and application thereof Download PDFInfo
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Abstract
The invention relates to a Halomonas sp LYX-3 separated from lead-zinc smeltery soil and application thereof in treatment of wastewater containing ethylene glycol monobutyl ether. Accession number of Halomonas sp.LYX-3: CGMCC No.22160. The Halomonas sp LYX-3 is used as follows: treating waste water containing ethylene glycol monobutyl ether.
Description
Technical Field
The invention relates to a Halomonas sp LYX-3 separated from lead-zinc smeltery soil and application thereof in treatment of waste water containing ethylene glycol butyl ether.
Background
Ethylene glycol monobutyl ether is an excellent organic solvent and has two functional groups with strong dissolving capacity of ether bond and hydroxyl. Wherein the ether bond has hydrophobicity and can dissolve hydrophobic compound; the hydroxyl group has good hydrophilicity, and can dissolve hydrophilic compounds. The above characteristics make butyl cellosolve widely used as solvent for nitrocellulose, spray paint, quick-drying paint, varnish, enamel and paint remover. It can also be used as fiber wetting agent, pesticide dispersant, resin plasticizer, organic synthetic intermediate, and reagent for measuring iron and molybdenum. It also improves the emulsifying properties, an auxiliary solvent for dissolving the mineral oil in the soap solution. Based on the wide application field of ethylene glycol butyl ether, the market demand is increasingly larger, but a large amount of harmful wastewater with high COD is generated in the production process of the ethylene glycol butyl ether, and the rho (COD) is as high as 30000-40000mg/L. Ethylene glycol butyl ether is inhaled and ingested orally and nasally, which may cause certain harm to human health, and may even be fatal, a high concentration (about 300-600 ppm) may cause headache, nausea and the like, and also may inhibit the central nervous system, a very high concentration may cause death, and animal experiments also find that ethylene glycol butyl ether may damage the reproductive system. Toxicity tests show that: LD 50 2500mg/kg (rat, oral); 1200ppm/4h (mouse, oral)).Therefore, the waste water containing ethylene glycol monobutyl ether must be effectively treated and discharged after reaching the standard.
Ethylene glycol monobutyl ether wastewater often has the characteristics of high organic matter content, complex water quality, poor biodegradability and the like, and causes great treatment difficulty. At present, common treatment methods for ethylene glycol monobutyl ether wastewater are an electrochemical method, a catalytic oxidation method, an incineration method and the like, and the treatment principle is that organic matters are directly converted into CO through a strong oxidation reaction 2 、H 2 Inorganic substances such as O and the like have the problems of harsh operating conditions, high running cost and the like, and influence on the wide application and popularization of the inorganic substances, so that an economic and efficient alternative treatment technology needs to be found, and foreign researches find that the microbial technology not only has good treatment effect, but also has low cost and no secondary pollution, so that the microbial technology is widely concerned, and related technical researches are few in China.
Currently, most of the known applications of halomonas (and its related genera) are functional microorganisms such as saline-alkali soil improvement, nitrogenous wastewater and biocontrol bacteria, and the like, and the halomonas (and its related genera) shows strong salt tolerance, nitrogen transformation and microorganism antagonism, and specifically comprises the following steps:
1. liu Hongsheng, xu Yingying, in dream of dream, zhang Qingying, wang Jianyan, tan Haochen, separation, identification and salt tolerance analysis of a saline and alkaline land of yellow river delta, university of anhui scholarly (nature science edition), 2015, 39 (4): 103-108;
2. wang Yue, a study of functional genes associated with nitrogen metabolism of the strain of alcaliomonas X3, the master thesis of shanghai ocean university, 2018;
3. liu Junhua, long Cong, lu Xiaoling, yang Qiao, cao Xia, liu Xiaoyu, jiao Binghua, identification and activity screening of a strain of halomonas from the east sea, chinese marine drugs, 2009, 28 (6): 5-10;
4. CN201210572369.0 of the invention, xanthomonas flava BJGMM-B45 and the application thereof, reported that: halomonas flava (Halomonas huanghegensis) BJGMM-B45 with the preservation number of CGMCC No:7022. the BJGMM-B45 can improve the tolerance of plants on the saline-alkali soil, and is a superior functional strain for improving the saline-alkali soil.
The research of Halomonas (Halomonas sp.) on the metabolism of the ethylene glycol butyl ether wastewater pollutant is not reported at home and abroad.
Disclosure of Invention
The invention aims to provide a strain, namely Halomonas (Halomonas sp.) LYX-3, which can be used for treating waste water containing ethylene glycol monobutyl ether and application thereof.
In order to solve the above technical problems, the present invention provides a Halomonas (Halomonas sp.) LYX-3, which has a deposit number: CGMCC No.22160.
The 16S rDNA gene sequence of the strain is shown in SEQ ID NO. 1.
The invention also provides the application of the Halomonas (Halomonas sp.) LYX-3: treating waste water containing ethylene glycol butyl ether; namely, removing the ethylene glycol butyl ether in the waste water containing the ethylene glycol butyl ether.
The Halomonas (Halomonas sp.) LYX-3 of the invention is separated from the soil of a lead-zinc smelting plant, and the preservation information is as follows:
the preservation name: halomonas sp, depository: china general microbiological culture Collection center, preservation Address: xilu No.1 Hospital No. 3, beijing, chaoyang, on Beijing, with a deposit number: CGMCC No.22160, preservation time 2021, 04 months and 09 days.
The bacterial colony characteristics of the strain are as follows: the bacillus is rough in surface, does not produce spores, has small capsules, is divided into two parts (monomer cells are transversely divided to form two daughter cells), is gram-negative, has the thallus size of (1.8-3.2) Mumx (0.6-0.8) Mum, and has circular colonies, convex and smooth surfaces, complete edges, lemon yellow and opaque on a solid culture medium.
The 16S rDNA gene sequence of the strain is shown in SEQ ID NO. 1.
The strain is obtained by screening collected soil of a certain lead-zinc smelting plant in Rizhou city of Hunan province, and according to a fatty acid map generated by qualitative and quantitative analysis of a Sherlock MIS software system on the strain LYX-3, a Library database is compared, so that the strain LYX-3 is preliminarily identified to be Halomonas (Halomonas sp.), and the similarity index SI (similarity index) is 0.98; the 16S rDNA sequencing identification method also proves that the strain is halomonas.
The invention also relates to the application of the Halomonas (Halomonas sp.) LYX-3 in the treatment of wastewater containing ethylene glycol monobutyl ether. Specifically, the strain is used for removing the butyl cellosolve in the waste water containing the butyl cellosolve. Inoculating the strain of the invention into waste water containing ethylene glycol butyl ether at 15-35 deg.C for 150-200 r.min -1 The culture is carried out for 0.5 to 2 days under the aerobic condition, and the ethylene glycol monobutyl ether in the wastewater can be effectively removed.
The waste water containing ethylene glycol butyl ether is waste water mainly containing ethylene glycol butyl ether, and the concentration of the ethylene glycol butyl ether in the waste water is 20-40000 mg.L -1 。
The strain can be used for treating waste water containing butyl cellosolve under aerobic condition, pH7.0, temperature 25-30 deg.C and OD 415nm 0.2, concentration of butyl cellosolve 19000 mg.L -1 (COD cr At 35000 mg.L -1 ) Rotational speed of 180r min -1 Culturing for 2 days, filtering, removing the butyl cellosolve with a removal rate of 99.99%, and reducing the concentration of butyl cellosolve to 1.9 mg.L -1 ，COD cr Reduced to 176 mg.L -1 Can reach the national comprehensive sewage discharge standard (GB 8978-1996) and enter the three-level standard (COD) of the urban sewage pipe network cr Is 500 mg.L -1 )。
The invention relates to a Halomonas (Halomonas sp.) LYX-3 separated from lead-zinc smeltery soil and application thereof in treatment of wastewater containing ethylene glycol monobutyl ether, which belong to the discovery for the first time.
In conclusion, the invention obtains the Halomonas (Halomonas sp.) LYX-3 for treating the wastewater containing the ethylene glycol butyl ether through screening, has the potential of efficiently treating the wastewater containing the ethylene glycol butyl ether, provides a technical basis for controlling the influence of the discharge of the wastewater containing the ethylene glycol butyl ether on the environment, and has wide application prospect.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 shows Halomonas sp LYX-3 (30000 times magnification);
FIG. 2 shows the results of 16S rDNA PCR amplification (LYX-3: strain LYX-3;
FIG. 3 is a phylogenetic tree of strains based on the 16S rDNA sequence.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
the culture medium is sterilized at 1.1 atm and 121 deg.C for 20min, which is common knowledge.
Example 1 screening and identification of degrading Strain
1 materials and methods
1.1 media and reagents
Enrichment culture medium: ethylene glycol monobutyl ether (5, 10, 20, 30 or 40 g) (gradual increase addition for acclimatization enrichment), caCl 2 ·6H 2 O 0.1g，MgCl 2 0.25g，K 2 HPO 4 1.5g，NH 4 Cl 1g, peptone 9g, H 2 O 500mL，pH＝6.5～7.5；
Basic culture medium: NH (NH) 4 NO 3 1.00g，MgSO 4 .7H 2 O 0.5g，(NH 4 ) 2 SO 4 0.5g，KH 2 PO 4 0.5g，NaCl 0.5g，K 2 HPO 4 1.5g，H 2 O 1000mL，pH4.0；
The basal medium was supplemented with 1.5% (w/v, 1.5g per 100 mL) agar and 40g.L -1 The ethylene glycol monobutyl ether is prepared into a corresponding solid basal culture medium;
pH7.0, ethylene glycol monobutyl ether concentration 19000 mg.L -1 (COD cr At 35000 mg.L -1 ) The waste water containing ethylene glycol monobutyl ether.
1.2 determination of treatment efficiency of waste water containing ethylene glycol monobutyl ether by bacterial strain
The single strain after purification is OD 415nm =0.2 inoculation to 100mL waste water containing butyl Ether glycol (pH7.0, butyl Ether concentration 19000 mg. L) -1 ，COD cr At 35000 mg.L -1 ) In a 250mL triangular flask, the culture medium without inoculation is used as a control, and the temperature is 30 ℃,180 r.min -1 The aerobic culture is carried out for 2 days on a constant temperature shaking table. After the culture is finished, filtering with a 0.20 mu m membrane to remove mycelium, and using the filtrate for corresponding butyl cellosolve and COD cr The content of ethylene glycol monobutyl ether is measured by gas chromatography, COD cr The value was determined using a COD meter.
Removal rate (%) = (control sample residual amount-treated sample residual amount) × 100/control sample residual amount.
1.3 dominant degradation Strain Breeding
1.3.1 sources of strains
Collecting soil of a certain lead-zinc smelting plant in shores of Hunan province.
1.3.2 isolation, purification and screening of degrading strains
10g of the lead-zinc smelting soil is taken from each part of lead-zinc smelting plant soil, and is respectively added into a 250mL triangular flask filled with 100mL of sterile liquid enrichment medium under the sterile condition, and 180 r.min is carried out at the temperature of 30 DEG C -1 After 7 days of cultivation on a shaker, the cells were transferred to the next enrichment medium in a 10% (vol%) inoculum size (diafenthiuron gradient of 5, 10, 20, 30 or 40g.L in this order) -1 ) And domesticating and culturing for 7 days under the same condition. Then the strain is transferred to the strain containing 80 g.L according to the inoculation amount of 10 percent -1 Continuously culturing in sterile liquid basic culture medium containing ethylene glycol monobutyl ether for 7 days, continuously inoculating for 2 times, and collecting the culture medium containing ethylene glycol monobutyl ether with concentration of 80 g.L -1 Taking 0.1mL of basic culture medium fermentation liquor in the basic culture medium, repeatedly carrying out solid basic culture medium plate streaking separation and purification until a single bacterial colony is obtained by screening, inoculating the pure bacterial colony on an inclined plane, and storing in a refrigerator at 4 ℃.
1.3.3 Strain identification
The strain identification adopts a Sherlock Microbiological Identity System (MIS) software system of MIDI company in America, and the system extracts and analyzes fatty acid of a single colony purified on a basic culture medium according to the operation specification of the MIDI company, qualitatively (type) and quantitatively (content) analyzes the fatty acid component of a microorganism and generates a fatty acid map, the generated map is compared with a database (Library), unknown strains are identified according to a similarity index SI (similarity index), and a certain microorganism can be basically determined if the similarity index SI is more than 0.9. The system is a microorganism identification system which is relatively quick and has rich bacterial banks, and has been widely applied (Wu Yuping, xu Jianming, wang Haizhen and the like. SherlockMIS system is applied to the research of soil bacteria identification. Soil bulletin, 2006, 43 (4): 642-647).
In addition, 16S rDNA identification method was also used for verification and comparison with Sherlock Microbiological Identity System (MIS). LYX-3 strain total DNA is used as template, 16S rDNA gene universal primer is used to make PCR amplification, the obtained amplified fragment is recovered and sequenced to determine its size, and then the sequencing result is compared with sequence in GenBank by using BLAST software to make homology comparison.
1.3.4 Observation of morphological characteristics of bacterial strains and determination of physiological and biochemical characteristics
Inoculating the strain in a solid culture medium, and observing morphological characteristics of the strain by using an electron microscope after 48 hours; taking the logarithmic phase of the growth of the purified strain to perform gram and crystal violet simple capsule staining and the like; the physiological and biochemical characteristics were determined according to the handbook of identification of common bacterial systems (Dongxu beads, cai Miaoying).
2. Results
2.1 isolation and screening of the strains
1 strain of bacteria which can effectively treat the waste water containing the ethylene glycol monobutyl ether is obtained through separation, purification and screening and is named as LYX-3, the degradability of other strains obtained in the screening process is declined to different degrees, and the removal rate of the ethylene glycol monobutyl ether is respectively 99.99 percent after aerobic culture for 2 days.
2.2 identification of the strains
2.2.1 basic morphology and physiological and biochemical characteristics of the Strain
The bacillus is bacillus, has rough surface, does not produce spores, has small capsule, has the division mode of binary division (two filial cells are formed by the transverse division of monomer cells), is gram negative, has the size of about 1.8-3.2 μm in length and 0.6-0.8 μm in width (as shown in figure 1), and has circular bacterial colony, convex and smooth surface, complete edge, lemon yellow color and opacity on a solid culture medium. Gram staining is negative, the reaction of the acetomethyl methanol generation test (v-p) is negative, the indole test is negative, gelatin cannot be liquefied, and other physiological and biochemical characteristics are shown in the table 1.
TABLE 1 physiological and biochemical characteristics of Halomonas
Note: + positive reaction; negative reaction
2.2.2 Strain identification
(1) Identification of counterweight by Sherlock MIS system
According to a fatty acid map generated by qualitative and quantitative analysis of the bacterium heavy fatty acid by a Sherlock MIS software system, comparing with a Library database, preliminarily identifying the bacterium as Halomonas (Halomonas sp.) and the similarity index SI (similarity index) as 0.98.
(2) Heavy verification of 16S rDNA identification method
DNA extraction results
PCR amplification was performed using the 16S rDNA gene universal gene using the recombinant total DNA as a template to obtain 1 fragment of about 1kb (FIG. 2). The amplified fragment is recovered and sequenced to confirm that the size of the amplified fragment is 1439bp (SEQ ID NO. 1).
II.16S rDNA Gene PCR amplification and sequence analysis
The sequence results are compared with the sequence in GenBank by using BLAST software to find that the strain LYX-3 has higher homology with the heavy salt monad (Halomonas sp.), the homology is close to 95 percent, and the genetic distance is close.
Combining the important physiological and biochemical properties, sherlock MIS (MIDI identification System) and 16S rDNA phylogenetic analysis, it can be seen that the bacterium is Halomonas sp.
3 conclusion
1 strain of high-efficiency LYX-3 which can be used for treating waste water containing ethylene glycol monobutyl ether is separated from the soil of a certain lead-zinc smelting plant in Hunan Zhou city and is identified as Halomonas (Halomonas sp.) through a Sherlock MIS system and 16S rDNA.
The preservation information is as follows:
the preservation name is: halomonas sp, depository: china general microbiological culture Collection center, preservation Address: xilu No.1 Hospital No. 3, beijing, chaoyang, on Beijing, with a deposit number: CGMCC No.22160, preservation time 2021, 04 months and 09 days.
Example 2 detection of treatment Performance of strains on waste water containing butyl cellosolve
1 materials and methods
1.1 media and reagents
Basic culture medium: NH (NH) 4 NO 3 1.00g，MgSO 4 .7H 2 O 0.5g，(NH 4 ) 2 SO 4 0.5g，KH 2 PO 4 0.5g，NaCl 0.5g，K 2 HPO 4 1.5g，H 2 O1000mL，pH4.0；
pH7.0, ethylene glycol butyl ether concentration 19000 mg.L produced in the production of ethylene glycol butyl ether -1 、COD cr At 35000 mg.L -1 The waste water containing ethylene glycol monobutyl ether.
1.2 treatment of waste water containing ethylene glycol monobutyl ether by bacterial strain
The purified single strain, halomonas sp, LYX-3 was measured as OD 415nm =0.2 inoculation to 100mL ethylene glycol butyl ether waste water (pH7.0, ethylene glycol butyl ether concentration 19000 mg. L -1 、COD cr At 35000 mg.L -1 Waste water containing ethylene glycol monobutyl ether) 250mL triangular flask, using the waste water containing the ethylene glycol monobutyl ether without inoculation as a reference, and controlling the temperature at 30 ℃ and 180 r.min -1 The aerobic cultivation is carried out on the constant temperature shaking table for 2 days. After the culture was completed, the removal rate of ethylene glycol monobutyl ether and CODcr by the strain was measured as described in example 1.
2. Results
The strain LYX-3 has pH of 7.0 and OD 415nm 0.2, rotation speed 180 r.min -1 Under the aerobic environment, the removal rate of butyl cellosolve is 99.99 percent, and the concentration of butyl cellosolve in the treated wastewater is 1.9 mg.L -1 ，COD cr Is 176 mg.L -1 Can reach the national comprehensive sewage discharge standard (GB 8978-1996) and enter the three-level standard (COD) of the urban sewage pipe network cr Is 500 mg.L -1 ) The invention shows that the isolated and screened Halomonas (Halomonas sp.) LYX-3 is a high-efficiency strain which can effectively treat the waste water containing the ethylene glycol butyl ether, and has certain application potential for controlling the influence of the waste water containing the ethylene glycol butyl ether on the environment.
Comparative experiments, in which LYX-3 was replaced by currently available halomonas (as described in Table 2 below), the assay was performed as described in example 2, and the results were compared with those of LYX-3 as described in Table 2 below.
TABLE 2
Therefore, the Halomonas sp LYX-3 discovered by the invention is an efficient strain which can effectively treat the waste water containing the butyl ether glycol, can efficiently remove the butyl ether glycol in the waste water, has certain application potential for controlling the influence of the waste water containing the butyl ether glycol on the environment, and other similar genera have no capacity or weak capacity.
Finally, it is also noted that the above-mentioned list is only a few 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> Zhejiang university
<120> Halomonas LYX-3 separated from the soil of the smeltery and its application
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<170> SIPOSequenceListing 1.0
<210> 1
<211> 1439
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
ggtggggacg ggtacctgca gtcgagcggc agcacgggga gcttgctccc tggtggcgag 60
cggcggacgg gtgagtaatg cataggaatc tgcccggtag tgggggataa cctggggaaa 120
cccaggctaa taccgcatac gccctacggg ggaaagcggg ggaccttcgg gcctcgcgct 180
atcggatgag cctatgtcgg attagctggt tggtgaggta acggctcacc aaggcgacga 240
tccgtagctg gtctgagagg atgatcagcc acactgggac tgagacacgg cccagactcc 300
tacgggaggc agcagtgggg aatattggac aatgggggca accctgatcc agccatgccg 360
cgtgtgtgaa gaaggccttc gggttgtaaa gcactttcag tgaggaagaa ggcctgcggg 420
ttaatagccg gcaggaagga catcactcac agaagaagca ccggctaact ccgtgccagc 480
agccgcggta atacggaggg tgcgagcgtt aatcggaatt actgggcgta aagcgcgcgt 540
aggtggcttg ataagccggt tgtgaaagcc ccgggctcaa cctgggaacg gcatccggaa 600
ctgtcaggct agagtgcagg agaggaaggt agaattcccg gtgtagcggt gaaatgcgta 660
gagatcggga ggaataccag tggcgaaggc ggccttctgg actgacactg acactgaggt 720
gcgaaagcgt gggtagcaaa caggattaga taccctggta gtccacgccg taaacgatgt 780
cgactagccg ttgggtgcct cgagcactta gtggcgcagt taacgcgata agtcgaccgc 840
ctggggagta cggccgcaag gttaaaactc aaatgaattg acgggggccc gcacaagcgg 900
tggagcatgt ggtttaattc gatgcaacgc gaagaacctt acctaccctt gacatcctcg 960
gaatctgccg gagacggcgg agtgccttcg ggaaccgagt gacaggtgct gcatggctgt 1020
cgtcagctcg tgttgtgaaa tgttgggtta agtcccgtaa cgagcgcaac ccctgtccct 1080
atttgccagc gattcggtcg ggaactctag ggagactgcc ggtgacaaac cggaggaagg 1140
tggggacgac gtcaagtcat catggccctt acgggtaggg ctacacacgt gctacaatgg 1200
caggtacaaa gggttgcaag acggcgacgt ggagctaatc ccataaagcc tgcctcagtc 1260
cggatcggag tctgcaactc gactccgtga agtcggaatc gctagtaatc gtgaatcaga 1320
atgtcacggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc atgggagtgg 1380
actgcaccag aagtggttag cttaaccttc gggagagcga tcaccacggg gttaccaag 1439
Claims (3)
1. Halomonas sp LYX-3, characterized by the accession number: CGMCC No.22160.
2. Use of the bacterium halopmonas (Halomonas sp.) LYX-3 of claim 1 wherein: is used for treating waste water containing ethylene glycol monobutyl ether.
3. Use of the bacterium halopoda (Halomonas sp.) LYX-3 according to claim 2, wherein: the method is used for removing the butyl glycol ether in the waste water containing the butyl glycol ether.
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