CN109880760B - Method for obtaining halophilic bacteria with high-salinity wastewater treatment function - Google Patents
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Abstract
The invention provides a method for obtaining halophilic bacteria with a high-salinity wastewater treatment function, and particularly relates to a method for transferring halophilic related genes into specific microorganisms.
Description
Technical Field
The invention relates to the field of sewage treatment, in particular to a method for obtaining halophilic bacteria with a high-salinity wastewater treatment function.
Background
In recent years, with the vigorous development of industrial industry in China, the discharge amount of industrial sewage is rapidly increased, with the diversified development of chemical synthesis process, most of high-salt and high-chlorine sewage which has high toxicity and has the effect of inhibiting or poisoning the growth of microorganisms is newly added in the industrial sewage, and the discharge of a large amount of standard sewage inevitably pollutes the surrounding environment and the receiving water body and endangers the surrounding sea area, thereby causing serious threat and influence on human life and production.
The biological strengthening technology is characterized in that microorganisms with specific functions are added into a biological treatment system to improve the treatment effect of the original treatment system, and the added microorganisms can be derived from the original treatment system.
Researchers at home and abroad apply the technology to the treatment of the refractory toxic and harmful substances in industrial wastewater, surface water and underground water or improve the wastewater treatment effect, and can obviously improve the microbial activity and the high-salinity wastewater treatment efficiency.
However, how to fully exert the potential of microorganisms, improve the treatment effect of refractory organic matters and inhibit the cytotoxicity of high-salt high-chlorine sewage on the microorganisms in the use process of the biological strengthening technology is a problem to be solved urgently in industrial production.
Disclosure of Invention
In order to solve the above technical problems, the present invention provides a method for obtaining halophilic bacteria having a high salinity wastewater treatment function.
The invention is realized by the following technical scheme:
a method for obtaining halophilic bacteria with high-salinity wastewater treatment function comprises transferring halophilic related genes into specific microorganisms.
Preferably, the microorganism includes, but is not limited to, bacillus, enterobacter, streptococcus, coccus, nitrobacteria, denitrifying bacteria, vibrio, actinomycetes, pseudomonas, flavobacterium, cyanobacteria, clostridium, globus, legionella, phytophthora, spirochete, devulcanium thermoaminogenes, mycobacterium, nocardia, micrococcus, cytophaga carbonate, lactic acid bacteria, mold.
More preferably, the microorganism is selected from the group consisting of bacillus subtilis and escherichia coli.
Further, the method comprises: obtaining total halophilic genes in a high-salt environment, constructing a halophilic gene library, primarily screening to obtain potential halophilic genes, transferring the potential halophilic genes obtained by primary screening into bacillus subtilis to obtain transgenic bacillus subtilis, and evaluating the salt tolerance of the transgenic bacillus subtilis.
Further, the halophilic related gene includes any one sequence selected from the group consisting of SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3.
Further, the halophilic related gene is shown as SEQ ID NO. 3.
Further, the halophilic related gene is present at least in a cell expressing the halophilic related gene.
Further, the cell may be a plant cell.
Further, the halophilic bacteria have tolerance and degradability to high-salt, high-chlorine and high-alkali sewage.
Further, the transformation includes, but is not limited to, heat shock method, injection method, genetic transformation method, gene editing method.
The invention has the following beneficial effects: after the transgenic bacillus subtilis obtained by the invention is added into the deep biochemical pool, the microbial life in the background sludge in the deep biochemical pool is not influenced, and a pilot test of 1 month proves that the transgenic bacillus subtilis provided by the invention has tolerance and degradability to high-salt, high-chlorine and high-alkali sewage, has a treatment function to the sewage with poor biodegradability, has stable single-bacterium performance, can be passaged through gene modification, and is particularly suitable for industrial scenes with deep sewage treatment requirements.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below.
Example 1: obtaining of Total halophilic Gene
The saline-alkali lake is a high saline-alkali environment in the nature, mostly an inland lake, can form a closed microbial ecological environment, a saline-alkali lake microbial community comprises algae, cyanobacteria, aerobic bacteria, anaerobic bacteria, facultative aerobic bacteria, archaea and the like, the archaea are important components of the saline-alkali lake microbial community, the archaea are suitable for the high saline-alkali environment, mostly belong to a high saline-alkali organic nutrition type, and are difficult to culture under conventional conditions due to the characteristics of osmotic pressure, cell membrane ion distribution, halophilic degree, poor nutrition and the like which are different from other microorganisms, and the archaea has the specificity on the physicochemical property, is different from the metabolic pathway of conventional microorganisms and has the potential of being applied to industrial industry, so the invention adopts a non-culture technology to explore the genetic diversity of the microorganisms including the archaea in the saline-alkali lake to obtain the microorganisms, in particular to halophilic related genes of archaea.
The sample is collected from China, namely Edinghu, the types of the sample comprise soil around the lake, salt shells on the surface of the lake, a water sample and mud at the bottom of the lake, and the sample is collected and then stored at 0 ℃. The annual precipitation of the Aiding lake is less than 20 mm, the annual average temperature is 14 ℃, the extreme high temperature reaches 48 ℃, and the earth surface temperature exceeds 80 ℃.
Mixing the soil around the lake, the salt shells on the lake surface, the water sample and the lake bottom soil sample, mixing 25g of the mixed sample with 50m of L buffer solution, adding into a dialysis bag, dialyzing for 12h to remove salt ions in the sample, after delaying, adopting a freeze-thaw method to crack cells and extract microbial total DNA, and purifying the DNA sample.
Example 2: construction of a salt-tolerant gene library
Sequencing the purified DNA, constructing salt lake halophilic gene library by using an Escherichia coli expression system (E.coli DH5a), randomly breaking genome DNA to 100bp, 150bp, 200bp, 250bp, 500bp, 1.0kb, 1.5kb and 2.5kb fragments, connecting the DNA fragments with a joint, performing PCR amplification to 100bp, 150bp, 200bp, 250bp and 500bp sequences, sequencing to obtain a short sequence library, constructing 1.0kb and 1.5kb large fragment libraries by using Cre-L ox library construction technology, connecting L oxP joints at two ends of the DNA large fragment, performing cyclization and sequencing, constructing insert fragments of 250bp, 500bp, 1.0kb and 1.5kb by genome construction steps, inserting the insert fragments into a DNA sequence through SO L EXA sequencing platform, inoculating the Escherichia coli inserted with exogenous gene to L B915 sequencing of different salt concentrations, performing sequencing, performing a homology test to obtain a strain with a NaCl strain with a homology test of no more than that the salt lake halophilic gene is inserted into a Nanjing Jinsi DNA sequence of 250bp, 200bp, 250bp, 1.0kb, inserting the 1.5kb and 1.5kb, inserting a strain DNA sequence into a genome sequencing platform, performing a strain DNA sequence, performing a strain amplification test to obtain a strain with a homology test to obtain a strain, performing a strain with a homology test result that the homology test is equal to obtain a strain, a strain with a homology test result that the homology test is equal to obtain a strain, a strain with a homology test that the concentration of a homology test that.
Example 3: transformation of halophilic genes into Bacillus subtilis
SA L, SA 7375 and SA L06 are codon modified to adapt to a Bacillus subtilis encoding system, the nucleotide sequence of the modified gene SA L c is shown in SEQ ID NO 1, the nucleotide sequence of SA L c is shown in SEQ ID NO 2, the nucleotide sequence of SA L c is shown in SEQ ID NO 3, the nucleotide sequences of SA 3941 c, SA L c and SA L c are synthesized by Nanjing Jinsry Biotech Limited, the 5 'end of the synthesized sequence is also connected with an NcoI enzyme cutting site, the 3' end is also connected with a SwaI enzyme cutting site, the synthesized nucleotide sequences of SA L c, SA L c and SA L c are respectively connected to a cloning vector pGEM-T (purchased from Promega), the operation steps are carried out according to a vector specification, and the recombinant cloning vectors pGEM-SA L c1, pGEM-SA L c5, AQEM-L c 2 c (AQEM-2 c-cDNA sequence is expressed by the codon 867 f promoter of the SP 867-RNA 867 f polymerase, the SP 867-RNA promoter is expressed by SP 867 f RNA-DNA polymerase, the SP 8653, the promoter expressed by SP 867 f, the SP 867-RNA polymerase promoter expressed by the SP 867 f, the SP 847, the promoter expressed by the SP 7, the promoter expressed by the expression of the promoter expressed by the.
Taking bacillus subtilis competent cells (sold in markets), transforming bacillus subtilis competent cells by recombinant cloning vectors pGEM-SA L c1, pGEM-SA L c5 and pGEM-SA L c6 by a heat shock method respectively, cloning by heat shock condition reference molecules, picking white colonies, culturing, extracting plasmids, carrying out enzyme digestion identification, and carrying out sequencing verification on positive clones, wherein the results show that the SA L1 c, SA L5 c and SA L6 c nucleotide sequences are correspondingly inserted into the recombinant cloning vectors pGEM-SA L c1, pGEM-SA L c5 and pGEM-SA L c6 respectively.
Example 4: salt tolerance test of transgenic bacillus subtilis
The above Bacillus subtilis into which the foreign gene was inserted was inoculated into L B liquid medium with 28% NaCl concentration, cultured at 30 ℃ for 5 days, observed for growth, and measured for salt content in the medium, the measurement results are shown in Table 1:
TABLE 1 measurement results of NaCl concentration in liquid medium varying with days
The result shows that the Bacillus subtilis transformed with the SA L1 c nucleotide sequence has no desalting effect, can not participate in high-salt metabolism in the metabolic process although tolerating a 28% high-salt culture medium and has vigorous growth vigor, the Bacillus subtilis transformed with the SA L5 c nucleotide sequence has vigorous growth vigor and stronger salt ion metabolic capacity in the later growth period and can meet the treatment condition of high-salt wastewater, and the Bacillus subtilis transformed with the SA L6 c nucleotide sequence has stronger salt metabolic capacity in the initial growth period but insufficient growth vigor, so that the high salt is obviously observed in the later growth period to inhibit the growth and development of the Bacillus subtilis.
Example 5: transgenic bacillus subtilis high-salinity wastewater treatment function pilot test
The pilot test process adopts a mixed activated sludge method, the pilot test equipment comprises a water pump, nutrient solution supply equipment, a deep biochemical tank, a third-stage sedimentation tank and recovery equipment, the water flow direction is that the effluent of a secondary sedimentation tank enters the deep biochemical tank through the water pump, the effluent of the deep biochemical tank enters the third-stage sedimentation tank, the recovery equipment recovers the sludge of the third-stage sedimentation tank and then mixes with the transgenic bacillus subtilis to flow back into the deep biochemical tank, the water inlet of the secondary sedimentation tank is connected with high-salt high-alkali wastewater, the biochemical indexes of the effluent of the secondary sedimentation tank are that COD is approximately equal to 120 mg/L3-N is approximately equal to 25 mg/L, the salt ion concentration is approximately equal to 0.28 mg/L, pH is equal to 8.9, the temperature is 35 ℃, the specification of the deep biochemical tank is 10m × 15m × 7.8.8 m, the deep biochemical tank contains local sludge, and the biochemical indexes of the effluent of the tertiary sedimentation tank are that COD is approximately equal to 89 mg/L when3-N ≈ 23 mg/L, salt ion concentration ≈ 0.257 mg/L, pH 8.5, temperature 32 ℃, detection period of one month.
Adding strains into a deep biochemical pool at first, wherein the addition amount of the strains is 1kg of strain dry powder, dissolving the strains in 1L nutrient solution, adding the strains into the deep biochemical pool together for acclimatization, keeping the dissolved oxygen at 2.4 mg/L, taking a water sample to detect whether the transgenic bacillus subtilis is colonized after running for two weeks, and finding out that the bacillus subtilis in a water sample in the deep biochemical pool is approximately 1.6 × 105CFU/m L, bacillus subtilis in local sludge is approximately equal to 2.3 × 108CFU/m L, the result shows that the transgenic bacillus subtilis has good colonization and good sludge growth state, after the exogenous bacteria are added, the sludge has no rejection reaction, the transgenic bacillus subtilis can be symbiotic with other bacteria, and after the operation for two weeks, the biochemical indexes of the effluent of the three-stage sedimentation tank are detected as that COD is approximately equal to 55 mg/L3-N ≈ 9.25 mg/L, salt ion concentration ≈ 0.117 mg/L, pH 7.5, temperature 32 ℃, total detectionThe period is one month.
According to the pilot test results, after the transgenic bacillus subtilis obtained by the invention is added into the deep biochemical pool, the microbial life in the background sludge in the deep biochemical pool is not influenced, and 1-month pilot test proves that the transgenic bacillus subtilis provided by the invention has tolerance and degradability to high-salt, high-chlorine and high-alkali sewage, has a treatment function to the sewage with poor biodegradability, has stable single-bacterium performance, can be subjected to passage through gene modification, and is particularly suitable for industrial scenes with deep sewage treatment requirements.
The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention, and it is therefore to be understood that the invention is not limited by the scope of the appended claims.
Sequence listing
<110> Yixing International environmental protection City science and technology development Co., Ltd
<120> a method for obtaining halophilic bacteria having high-salinity wastewater treatment function
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ttcgattcag gcagcctttg atccgatgaa tcctatgtga cacagtgcaa tttaacctgt 120
taaaaggaga tctcaagcat gccctttttc cggacttaga cgatcagata cctacaccat 180
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attcacgtgg gattaaacga cagtcgctac agaacgatct gcttgccaga atagaggctc 300
gcgggggtta cgcacttcct tcaccacaac gtttcctgga gtggatgcag gggagagatg 360
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tactgcgagt ggattgaaat ctgcacgtcg cgccgtcagt aaatataaca cagacgacca 480
ttgacttccg aagtcacttg caggagttcc tcctcatatg ctcctcgacc agaggtgaga 540
cccaggtctc cttgtttcct ggacgtgcac atcagccgtg gtacaacatt cacgtacatt 600
gtccttctgt gatgccgtcc attctaatgg cctagccact cctcacgcac gacgtgggct 660
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tcgttcctct gctgatctct tagctaattg ggctgattct tgcagtagtt gaatataacg 1020
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cacgccaatg ccgatcgggg tggagagaaa gacatatccg gagggctggc gctgtgcccg 1140
cgttagggcg tagtccctaa ttctcctgag cctattccgc atcacacgca tcgactgcaa 1200
attgatttca gacggcagga caatcagagt aatcagcctc gcaatttcgt cctattgtct 1260
aagccaggtg atagaggtga 1280
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gcccgacaaa cttccgatgc tgatccccgg atagcctcgc taaggcgctt gggatcaagt 60
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cacgaatgta gtatccttgg ctgttccctg aggacgggga gctatgccat gcctcacgtg 180
attatttaat taacatcatt ttggtcgttt gggtgaaagg ctcactgcag gaacttgatc 240
acctagaata atggcataat gggccgcttt cgtatatccg gtaaccgtcc ttaaaacatc 300
ccttccttta ggcacgttag agctagacgc gatttttagc cagatgacct atctaccacc 360
gcctccgtaa cagggggcag cgaacagacc acccaccgtt aaaatgtgtc tcccgacgaa 420
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aggtttcagt ggctcattac tactaaggat tcacgtatct gaacttagcg cttggccaac 1260
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taatatacac actagatgtc gggccattcc aatgtcaggt tacaaccacc ccggtaaatg 180
ggtaatactt cccacgcgca gctagatccg gcatttgccc ataatcaaaa gaggtgcgct 240
ttattccctt caggacttcc aaagaggctc gcgggtcctg aactcctaat cctgtatagt 300
cctcaagctg cagtctagat aacgtaagga ctcggcaaaa tcgggtagga aatttagcgt 360
ttcagcatgt atgtagtcgc gccgtttata caagacagcc aagacgctcc ttccgcgcct 420
attaaaggac gctagctaca tcgctggctt aatgtttggc ggatggaaaa ctacttagct 480
tataggaggc tcaattacaa atctattcga tacgatgggc tttttagtgc agccaggctg 540
gtcctcgcag gctgaaactt aatcagtctt tgttctcagg cgcctgccgg aacccaatgg 600
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accaggtcgg gtcttgttat tagatgaaat gctaactaag cgtacacggc actgggtacc 720
cgcgtccttt tagcgggatc tctccctgca gccctccaag tggacttata agagtggcct 780
ctgttatcgt ctgatggcat atgaccagac ggttacagga tatgcggctg agacacgacg 840
ctctctagtt atatatttga cgagcgaatg cgtgttacac cactagtcgt aacactcccc 900
gcactatagc gagtaattga ttgtcctcga gcgaggtaag taaggatggc ctagcaaccg 960
agaagcatct gggcaaagtt ggaacaaata gtagaacaac aacatattat tgctactgat 1020
aaagaacttt gtgccgattc actccctctg gacagagtta atccctctgg ggaagcccaa 1080
atcattatta tgcccaacgg tcctgatgta acttcgcggt gtgaggtgtg gtagtaacca 1140
ggtaatgcca cgcgcgtcta aaatcctctc cgatcgaagc gaggagccgt acagtactga 1200
gatctcacat tcacggagat caaacctagt gagctccacc attcaccgta gaatgtgcgt 1260
ccatgactcc gctgttctta gttgtccggg tgttgcttga gagtat 1306
Claims (5)
1. A method for obtaining halophilic bacteria with high-salinity wastewater treatment function, which is characterized by comprising transferring halophilic related genes into specific microorganisms;
the microorganism is selected from bacillus subtilis;
the halophilic related gene comprises any one sequence selected from SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3.
2. The method of claim 1, wherein the method comprises: obtaining total halophilic genes in a high-salt environment, constructing a halophilic gene library, primarily screening to obtain potential halophilic genes, transferring the potential halophilic genes obtained by primary screening into bacillus subtilis to obtain transgenic bacillus subtilis, and evaluating the salt tolerance of the transgenic bacillus subtilis.
3. The method of claim 1, wherein the halophilic related gene is shown in SEQ ID NO. 3.
4. The method of any one of claims 1 to 3, wherein the halophilic bacteria are tolerant to and degradable by high salinity, high chlorine, high alkalinity wastewater.
5. The method of claim 1, wherein said transformation comprises but is not limited to heat shock method, injection method, genetic transformation method, gene editing method.
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