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. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Isolation and characterization of A.calcoaceticus FL04
Experimental wastewater and sludge: secondary sedimentation tank sludge of sewage treatment plant of Makou town of Hanchuan city, Hubei province, and water outlet of printing and dyeing mill.
Enrichment culture medium: 1g/L phthalic acid, 0.1g/L benzidine and the balance of water, pH7.0
Screening a culture medium: 1g/L phthalic acid, 0.1g/L aniline, 0.5g/L yeast extract powder, 0.2g/L MgSO4 7H2O,0.1g/L CaCl2,1g/L(NH4)2SO4,1g/L K2HPO4,0.1g/L NaCl,0.02g/L FeCl3The balance being water, pH 7.0;
solid screening culture medium: 1g/L phthalic acid, 0.1g/L aniline, 0.5g/L yeast extract powder, 0.2g/L MgSO47H2O,0.1g/L CaCl2,1g/L(NH4)2SO4,1g/L K2HPO4,0.1g/L NaCl,0.02g/L FeCl315g/L agar powder and the balance of water, and the pH value is 7.0;
strain separation:
adding 90mL of enrichment medium into a 250mL triangular flask, then respectively adding sludge in a secondary sedimentation tank of a sewage treatment plant and 10g of soil near a water outlet of a printing and dyeing mill, sealing, placing on a shaking table at 37 ℃, and performing shaking culture at 150rpm for 24 hours to obtain an enrichment culture solution;
and (3) putting 100mL of screening culture medium into a 250mL triangular flask, respectively adding 1mL of enrichment culture solution, sealing, placing on a 37 ℃ shaking table, and performing shake culture at 150rpm for 48h to obtain a target culture. After being diluted in a gradient way, the target culture is coated on a solid screening culture medium (liquid screening culture medium +15g/L agar powder) plate, is kept stand and cultured for 48 hours at 37 ℃, and a single colony is picked, so that the required bacterial strain can be obtained.
Strain characterization and identification:
the colonies are semitransparent white on an LB culture medium, have smooth edges, are irregular and round, are glossy and are easy to pick; the thallus is rod-shaped, gram-negative bacteria, the optimal production pH is 6.8, the optimal growth temperature is 34-37 ℃, and the optimal osmotic pressure (NaCl%) concentration is 1.2%.
The selected single colony is preserved and has the 16S rDNA (sequence is provided) gene sequence length of 1466bp, and the 16S rDNA of the acinetobacter calcoaceticus FL04 is compared with a GenBank database by a BLAST analysis method, so that the genetic relationship between the acinetobacter calcoaceticus KW3 and the acinetobacter calcoaceticus is the closest, and the homology is as high as 99%. Phylogenetic evolution analysis was performed on both of them using MEGA program and phylogenetic trees were drawn as shown in FIG. 1. The strain is named acinetobacter calcoaceticus FL04 with a deposit number (M2019461).
Application of acinetobacter calcoaceticus FL04 in treatment of benzene-containing printing and dyeing wastewater
Taking the effluent from the hydrolytic acidification tank of the printing and dyeing mill (CODCr is 800 +/-10.2 mg/L), putting the effluent into 2000mL triangular bottles, wherein each bottle contains 1000mL, and experimental groups are inoculated with the acinetobacter calcoaceticus powder (the viable count concentration of the powder is 2 multiplied by 10)9cfu/g) of the component(s) added to the printing and dyeing wastewater to a final concentration of not less than 1X 106cfu/mL (i.e., 0.5g of fungal powder per liter of wastewater), the control group was not inoculated with microorganisms and was treated with Acinetobacter calcoaceticus NCIMB9871 from Shanghai Biotech, Inc. In the treatment process, small aeration equipment is used for aeration, so that the dissolved oxygen in the water is not less than 2 mg/L. And (3) performing parallel three groups of experiments in each group, and determining the CODCr and the aniline content of the wastewater after culturing for 72 hours.
Specifically, the acinetobacter calcoaceticus FL04 powder used in the experimental group is obtained by the following method: inoculating acinetobacter calcoaceticus FL04 into a fermentation culture medium, sealing, placing in a shaking table at 30-37 ℃, performing shake culture for 40-48h to obtain acinetobacter calcoaceticus FL04 bacterial liquid, adding bentonite as a carrier, and performing spray drying to obtain acinetobacter calcoaceticus FL04 bacterial powder.
Specifically, the fermentation culture comprises 10g/L beef extract, 0.5g/L yeast extract powder, and 0.2g/LMgSO4 7H2O、0.1g/L CaCl2、1g/L(NH4)2SO4、1g/L K2HPO4,0.1g/L NaCl,0.02g/L FeCl3The balance of water, and the pH value is 6.8-7.0.
Specifically, the printing and dyeing wastewater is added with the initial concentration, the culture temperature and the oxygen concentration after aeration of the Acinetobacter calcoaceticus FL04 bacterial liquid, and the COD (chemical oxygen demand) in the wastewater treated by the Acinetobacter calcoaceticus FL04CrAnd aniline concentrations are shown in Table 1, to give examples 1-6 and comparative examples 1-2. COD of dyeing wastewater before treatmentCr800 +/-10.2 mg/L, 0.88 +/-0.02 mg/L of aniline substance, and 1.15 multiplied by 10 for the initial concentration of the treated acinetobacter calcoaceticus FL04 bacterial liquid of comparative example 1 provided by the invention5cfu/mL, comparative example 2 used Acinetobacter calcoaceticus NCIMB9871, supplied by Shanghai Biotech, Inc. Comparative example 3 no microorganism was added.
Finally, the COD in the treated wastewater is investigatedCrAnd aniline concentration, CODCrThe measurement of (2) is carried out by the method of HJ828-2017, and the measurement of anilines in water is carried out by GB/T11889-1989.
The results in Table 1 show that by selecting the acinetobacter calcoaceticus FL04 provided by the invention, the culture time of wastewater treatment and the oxygen concentration after aeration are controlled, and the concentrations of chromium metal COD and aniline in wastewater can be effectively controlled and reduced. Example 4 is the best for treating printing and dyeing wastewater relative to the comparative example.
TABLE 1 treatment results of dyeing wastewater
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> university of agriculture in Huazhong
<120> acinetobacter calcoaceticus and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1466
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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attgaacgct ggcggcaggc ttaacacatg caagtcgagc ggagtgatgg tgcttgcact 60
atcacttagc ggcggacggg tgagtcatgc ttaggaatct gcctattagt gggggacaac 120
atttcgaaag gaatgctaat accgcatacg tcctacggga gaaagcaggg gatcctcgga 180
ccttgcgcta atagatgagc ctaagtcgga ttagctagtt ggtggcgtaa aggcctacca 240
agtcgacgat ctgtagcggg tctgagagga tgatccgcca cactgggact gagacacggc 300
ccagactcct acgggaggca gcagtgggga atattggaca atgggcgcaa gcctgatcca 360
gccatgccgc gtgtgtgaag aaggccttat ggttgtaaag cactttaagc gaggaggagg 420
ctactgaagt taataccttc cgatagtgga cgttactcgc agaataagca ccggctaact 480
ctgtgccagc agccgcggta atacagaggg tgcaagcgtt aatcggatta actgggcgta 540
aagcgcgcgt aggcggctaa ttaagtcaaa tgtgaaatcc ccgagcttaa cttgggaatt 600
gcattcgata ctggttagct agagtgtagg agaggatggt agaattccag gtgtagcggt 660
gaaatgcgta gagatctgga ggaataccga tggcgaaggc agccatctgg cctaacactg 720
acgctgaggt gcgaaagcat ggggagcaaa caggattaga taccctggta gtccatgccg 780
taaacgatct ctactagccg ttggggcctt tgaggcttta gtggcgcagc taacgcgata 840
agtagaccgc ctggggagta cggtcgcaag actaaaactc aaatgaattg acgggggccc 900
gcataagcgg tggagcatgt ggtttaattc gatgcgacgc gaagaacctt acctggcctt 960
gacatagtaa ggactttcca gagatggatt ggtgccttcg ggaacttaca tacaggtgct 1020
gcatggctgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1080
ccttttcctt atttgccagc gagtaatgtc gggaacttta aggatactgc cagtgacaaa 1140
ctggaggaag gcggggacga cgtcaagtca tcatggccct tacggccagg gctacacacg 1200
tgctacaatg gtcggtacaa agggttgcta cctagcgata ggatgctaat ctcaaaaagc 1260
cgatcgtagt ccggattgga gtctgcaact cgactccatg aagtcggaat cggtagtaat 1320
cgcggatcag aatgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac 1380
catgggagtt tgttgcacca gaagtaggta gtctaaccgc aaggaggacg cttaccacga 1440
tgtggccgat gactggggtg ggctca 1466