CN114621903B - Degradation strain for treating medical wastewater and application of degradation strain in bio-enhancement treatment - Google Patents
Degradation strain for treating medical wastewater and application of degradation strain in bio-enhancement treatment Download PDFInfo
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Abstract
The invention discloses a degradation strain for treating medical wastewater and application thereof in biological strengthening treatment, wherein the degradation strain is stenotrophomonas parvula (Stenotrophomonas pavanii), the strain is named JG-1 and is preserved in China general microbiological culture Collection center (China center) for 12 months of 2021, and the strain preservation number is: CGMCC No.24036. The strain is applied to the medical wastewater through a biological strengthening technology, so that the medical intermediate or sulfanilamide medicines in the industrial wastewater or soil can be effectively removed, and the problems of strong inhibition effect and low degradation efficiency of the medical intermediate and the sulfanilamide medicines on functional microorganisms are solved.
Description
Technical Field
The invention belongs to the technical field of biological wastewater treatment, and particularly relates to a degradation strain for treating medical wastewater and application of the degradation strain in biological strengthening treatment.
Background
The production of pharmaceutical intermediates is an indispensable link in the production of pharmaceuticals, meaning that various pharmaceutical intermediates need to be produced. At present, more than 2000 raw materials and intermediates matched with medicines are needed in China every year, and the demand is more than 250 ten thousand tons. With the production of the medical intermediate, various different waste water can be generated, and compared with common waste water, the production waste water of the medical intermediate has the characteristics of high COD, high salinity, complex components, poor biodegradability, high biotoxicity and the like.
Acetanilide (N-AA) is an important organic synthesis intermediate thereof and is widely used in the synthesis process of medicines. Mainly antibiotics such as Oxytetracycline (OTC), doxycycline (DOX) synthetic raw materials; also non-steroidal anti-inflammatory salicylic acids (aspirin), anilines (phenacetin, paracetamol), pyrazolones (phenylbutazone, oxybenzone) and other organic acids (ibuprofen). P-aminobenzenesulfonamide (p-ASA) is an organic compound synthesized by taking acetanilide as a raw material, and is an intermediate synthesized by sulfonamides such as Sulfanilamide (SA), methanesulfonylaniline (MSA), sulfaguanidine (SGD), sulfaisoxazole (SIX), sulfacetamide (SCM), sulfadiazine (STZ), sulfadiazine (SDZ), sulfapyridine (SPD), sulfamethyidine (SMZ), sulfadimine (SDM), sulfametadoxine (SMT) and the like. For production process reasons, a large amount of intermediates remain in industrial wastewater and are a major source of chemical oxygen demand for industrial wastewater. In addition, industrial wastewater is difficult to biologically treat because of the strong inhibition effect of the intermediates and the medicaments on organisms.
The biological strengthening technology is to add microorganisms with specific functions and the like into a traditional wastewater biological treatment system, so as to improve degradation efficiency of refractory substances. The biological strengthening technology can exert the function of microorganisms to the greatest extent and practically improve the processing capacity of a biochemical system. Aiming at the medical wastewater difficult to degrade, the running stability and the treatment effect of the sewage treatment facility can be greatly improved by adding the special degrading microorganism. Therefore, the research of treating the organic wastewater difficult to degrade by adopting the biological strengthening technology has received general attention from students at home and abroad. Compared with other treatment methods, the biological method has simple operation, low cost and no secondary pollution, but the activity of the microorganisms which are not domesticated during the biological method treatment is obviously inhibited, thereby seriously affecting the efficiency of wastewater treatment. In order to improve the treatment effect, it is necessary to domesticate and culture a highly efficient degradation strain which can survive in wastewater having biotoxicity and can tolerate high salt. Therefore, the high-efficiency degradation strain capable of degrading the acetanilide and the p-aminobenzene sulfonamide simultaneously is screened out, and has important environmental benefit and good industrial application prospect for biological treatment of pharmaceutical wastewater.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a degradation strain for treating medical wastewater and application thereof in biological strengthening treatment, and medical intermediates and sulfonamides in industrial wastewater are effectively removed, so that the problems of strong inhibition effect and low degradation efficiency of the medical intermediates and the sulfonamides on functional microorganisms are solved.
In order to achieve the above object, the present invention adopts the following technical scheme:
the degrading strain for treating the medical wastewater is stenotrophomonas parvula (Stenotrophomonas pavanii), the strain is named JG-1 and is preserved in China general microbiological culture Collection center (China center for type culture collection) at 12 months of 2021, and the strain preservation number is: CGMCC No.24036.
A microbial inoculum produced by the degradation strain JG-1.
The preparation method of the microbial inoculum comprises the following specific steps:
s1, inoculating a strain JG-1 stored in an inclined plane into an LB solid medium for activation culture;
s2, picking single colonies of the activated strain JG-1, inoculating the single colonies into an LB liquid culture medium, and culturing the single colonies in an oscillating manner until a logarithmic growth phase is reached, wherein the single colonies are used as seed liquid;
s3, inoculating the seed solution into a fresh LB culture medium, continuously performing expansion culture, collecting the culture solution, centrifuging, discarding the supernatant to collect thalli, and finally re-suspending thalli cells with sterile water to obtain the microbial inoculum.
Preferably, in the aforementioned step S1, the composition of the LB solid medium is: 10g/L peptone, 5g/L, naCl g/L yeast extract, 2% agar; the culture conditions are as follows: the temperature is 25-35 ℃ and the time is 24-48 h.
Preferably, in the aforementioned step S2, the components of the LB liquid medium are: 0.2g/L of p-aminobenzenesulfonamide, 0.2g/L of acetanilide, 10g/L of peptone, 5g/L of yeast extract and 10g/L of NaCl; the culture conditions are as follows: the temperature is 25-35 ℃, the rotating speed is 160-200 r/min, and the time is 12-24 h.
Preferably, in the aforementioned step S3, the fresh LB medium comprises the following components: 10g/L peptone, 5g/L yeast extract and 10g/L NaCl; the culture conditions are as follows: the temperature is 25-35 ℃, the rotating speed is 160-200 r/min, and the time is 24-48 h.
An application of degradation strain in degrading medicine intermediate or sulfanilamide medicines.
Application of a microbial inoculum in degrading medical intermediates or sulfonamides.
Preferably, the medical intermediate is one or two of acetanilide and sulfanilamide, and the sulfanilamide is one or more of sulfanilamide, sulfapyridine and sulfaisoxazole.
The invention has the advantages that:
(1) The invention provides a P.pajami strain JG-1 capable of degrading medical intermediate production wastewater, which is applied to medical wastewater by a biological strengthening technology, can effectively remove medical intermediates of acetanilide and sulfanilamide in industrial wastewater, and solves the problems of strong inhibition effect and low degradation efficiency of the medical intermediates and sulfanilamide drugs on functional microorganisms;
(2) The strain JG-1 can degrade sulfonamide, sulfapyridine, sulfaisoxazole and other sulfanilamide drugs and antibiotic terramycin, can be prepared into microbial inoculum suspension, is applied to degradation and removal of sulfanilamide drug pollutants in different environmental media such as sulfanilamide pharmaceutical enterprise wastewater, medical wastewater or soil, and has good industrial application prospect and environmental benefit.
Drawings
FIG. 1 is a colony morphology of the strain JG-1 of the present invention;
FIG. 2 is a gram-stained photograph of the strain JG-1 of the present invention;
FIG. 3 shows the degradation effects of the strain JG-1 of the present invention on acetanilide and sulfanilide;
FIG. 4 is the effect of NaCl concentration on the degradation effect of strain JG-1 according to the present invention;
FIG. 5 shows the degradation effect of the strain JG-1 of the invention on different substrates;
FIG. 6 shows the effect of the strain JG-1 of the present invention on the treatment of medical wastewater.
Detailed Description
The invention is described in detail below with reference to the drawings and the specific embodiments.
Example 1
Separation, screening and identification of degradation strain JG-1:
1. separation and screening of degradation strain JG-1
Taking supernatant of activated sludge sediment of industrial wastewater treatment of a pharmaceutical enterprise of 2 mL, inoculating into 100mL of inorganic salt culture medium, and performing proliferation culture at 30 ℃ and 160r/min for 48h. Inoculating 5mL of enrichment solution into 100mg/L of acetanilide and 100mg/L of p-aminobenzenesulfonamide in an inorganic salt culture medium, and shake culturing at 30 ℃ and 160r/min for 5d; and 5mL of enrichment culture solution is inoculated into the new inorganic salt culture solution each time, and shaking culture is continued, and the concentration of acetanilide and p-aminobenzenesulfonamide is increased by 100mg/L each time. After 6-7 times of inoculation, the concentration of the acetanilide and the para-aminobenzene sulfonamide in the enrichment culture solution reaches 400mg/L, and the culture solution is transferred into the enrichment culture solution with the concentration of 500mg/L of the acetanilide and the para-aminobenzene sulfonamide again for growth. The enrichment culture solution is gradually diluted and then evenly coated on LB solid culture medium, cultured at 30 ℃ and observed for colony morphological characteristics. Pure bacterial colonies on a flat plate are selected and inoculated into an inorganic salt culture medium with 500mg/L of p-aminobenzenesulfonamide and 500mg/L of acetanilide for 3 days at 30 ℃ and 160r/min, degradation effects are detected, and dominant bacterial strains with high degradation rate are screened out, drawn on an LB culture medium for purification and preservation, and further research is carried out.
Wherein, the inorganic salt culture medium comprises the following components: caCl (CaCl) 2 0.02g/L、(NH 4 ) 2 SO 4 1.0g/L、NaCl 0.5g/L、FeCl 3 0.05g/L、K 2 HPO 4 1.0 g/L、KH 2 PO 4 1.0g/L、NH 4 NO 3 1.0g/L and distilled water 1L, pH 7.0, 2% agar was added to the solid medium, and the medium was sterilized at 121℃for 30 minutes.
The LB medium comprises the following components: 10g/L peptone, 5g/L, naCl g/L yeast extract, 2% agar and 1L distilled water, pH 7.0, and sterilizing the culture medium at 121deg.C for 30min.
2. Identification of degradation Strain JG-1
Through enrichment culture, a strain capable of efficiently degrading acetanilide and p-aminobenzenesulfonamide is screened out, and is named JG-1. The strain is identified by 16S rDNA sequence (see sequence table in detail) and the strain JG-1 is determined to be P.pastoris oligotrophic monadStenotrophomonas pavaniiAnd deposited in China general microbiological culture Collection center (China center) for type culture collection, with a culture collection number of 2021, 12 th month, 6 th month: the bacterial colony morphology of the strain in the LB solid medium is shown in figure 1, and the bacterial colony is round, neat in edge, convex, gray, smooth, moist, semitransparent and gram-negative (see figure 2).
Example 2
Degradation effect of degradation strain JG-1:
the single colony of the degradation strain JG-1 obtained by screening in the example 1 is selected and inoculated in 100mL of LB liquid medium, the culture is carried out at 30 ℃ and 160r/min until the logarithmic phase, the obtained culture solution is centrifuged at 4 ℃ and 8000r/min for 3 min, the supernatant is removed to collect thalli, and the thalli are resuspended in sterile water to be used as bacterial suspension.
Respectively preparing inorganic salt liquid culture mediums with the concentration of acetanilide and p-aminobenzenesulfonamide of 500mg/L, inoculating the obtained bacterial suspension into the inorganic salt culture mediums in a volume ratio of 2%, carrying out constant-temperature shaking culture at 30 ℃ and 160r/min, sampling at regular time, and measuring the concentration of the acetanilide and the p-aminobenzenesulfonamide in the culture liquid.
The strain JG-1 can grow by taking acetanilide and p-aminobenzenesulfonamide as carbon sources, and according to the figure 3, the degradation rate of p-aminobenzenesulfonamide (p-ASA) can reach 60 percent at 36 hours, and the degradation rate of acetanilide (N-AA) is 70 percent; 48 The degradation rate of h reaches about 90%, and the result shows that the strain JG-1 can rapidly and efficiently degrade the acetanilide and the p-aminobenzenesulfonamide.
Example 3
Effect of NaCl concentration on strain JG-1 degradation:
inorganic salt liquid culture mediums with different NaCl concentrations are respectively prepared, so that the NaCl concentrations are respectively 1g/L, 5g/L, 10g/L, 15g/L and 20g/L, and the pH value of the culture medium is adjusted to 7.0. The bacterial suspension in example 2 was inoculated into an inorganic salt culture medium containing acetanilide and p-aminobenzenesulfonamide at a volume ratio of 2%, cultured for 48 hours at 30℃under 160r/min with shaking, the concentration of acetanilide and p-aminobenzenesulfonamide in the culture medium was detected and the degradation rate was calculated, and the tolerance of the strain JG-1 in environments of different salt concentrations was verified.
According to the graph 4, in the NaCl concentration range of 1-20 g/L, the strain JG-1 has high degradation efficiency on acetanilide and sulfanilide, the degradation rate is over 70%, and the strain JG-1 has good salt tolerance, thereby providing favorable conditions for treating high-salinity industrial wastewater.
Example 4
Degradation effects of strain JG-1 on different substrates:
the tetracycline antibiotics terramycin (OTC) and Doxycycline (DOX) synthesized by using acetanilide as raw materials and sulfanilamide drugs Sulfanilamide (SA), sulfapyridine (SPD) and Sulfaisoxazole (SIX) synthesized by using sulfanilamide as intermediates are selected, and the degradation effect of the strain JG-1 on downstream products of the pharmaceutical intermediates is verified.
Inorganic salt culture mediums of OTC, DOX, SA, SPD and SIX were prepared at a final concentration of 100mg/L, respectively, and the pH of the culture medium was adjusted to 7.0. The bacterial suspension in example 2 was inoculated into the above-mentioned different inorganic salt media at an inoculum size of 5% (V/V) by volume, cultured in a constant temperature shaking oven at 30℃and 160r/min, and after 5 days, the substrate concentrations in the media were measured and the degradation rate was calculated. The degradation effect of the strain JG-1 on different substrates is shown in FIG. 5. As can be seen from FIG. 5, strain JG-1 can effectively degrade SA, SPD, SIX and has a certain degradation capacity for OTC, but cannot degrade DOX.
Example 5
Preparation of microbial agent:
the strain JG-1 preserved on the inclined plane is inoculated into LB solid culture medium (the components of the LB solid culture medium are peptone 10g/L, yeast extract 5g/L, naCl g/L and 2% agar) and then is subjected to activation culture at a constant temperature of a 30 ℃ incubator for 24h. Then, single colony of the activated strain JG-1 is selected and inoculated into an LB liquid culture medium (the formula of the LB liquid culture medium is that N-AA 0.2g/L, p-ASA 0.2g/L, peptone 10g/L, yeast extract 5g/L and NaCl 10 g/L), and the culture is carried out at the constant temperature of 30 ℃ and 160rpm, and the culture is carried out until the logarithmic phase is reached and the culture is used as seed liquid; then inoculating 10% (V/V) of seed solution into fresh LB culture medium (the components of the fresh LB culture medium are peptone 10g/L, yeast extract 5g/L and NaCl 10 g/L), continuously expanding culture, collecting culture solution, centrifuging at 4 ℃ and 8000r/min for 3 min, discarding supernatant, collecting thalli, washing thalli with sterile water, resuspension, centrifuging again, removing supernatant, repeating the steps for 3 times to obtain washed thalli, and finally resuspension thalli with sterile water to obtain microbial inoculum.
Example 6
The bacterial strain JG-1 has the treatment effect on medical wastewater:
the obtained stenotrophomonas pajami JG-1 bacterial liquid is added into simulated medical wastewater W1 and W2 according to the inoculation amount of 5%, activated sludge is inoculated in a control group, and the activated sludge is sampled at 0 d, 1 d, 2 d, 3 d, 4 d, 5d and 6 d respectively, and the degradation rate is detected and calculated, and the result is shown in figure 6.
W1 wastewater composition: acetamine 500mg/L, terramycin 50mg/L, sodium chloride 1000mg/L, caCl 2 0.02g/L、(NH 4 ) 2 SO 4 1.0g/L、FeCl 3 0.05g/L、K 2 HPO 4 1.0 g/L、KH 2 PO 4 1.0g/L、NH 4 NO 3 1.0g/L and distilled water 1L.
W2 wastewater composition: 200mg/L acetanilide, 200mg/L sulfanilamide, and sodium chloride 1000mg/L, caCl at a concentration of 20 mg/L sulfanilamide and sulfapyridine 2 0.02g/L、(NH 4 ) 2 SO 4 1.0g/L、FeCl 3 0.05g/L、K 2 HPO 4 1.0 g/L、KH 2 PO 4 1.0g/L、NH 4 NO 3 1.0g/L and distilled water 1L.
As shown in FIG. 6, the conventional sludge has no effect on the sludge, and after the P.pastoris (JG-1) is added, the pollutant in the wastewater can be effectively removed, the removal rate is about 80%, and the sludge can be applied to medical wastewater treatment.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be appreciated by persons skilled in the art that the above embodiments are not intended to limit the invention in any way, and that all technical solutions obtained by means of equivalent substitutions or equivalent transformations fall within the scope of the invention.
Sequence listing
<110> Jiangsu Pop technologies Co., ltd
<120> degradation strain for treating medical wastewater and application thereof in bio-enhancement treatment
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1449
<212> DNA
<213> stenotrophomonas pajami (Stenotrophomonas pavanii)
<400> 1
ggcaatggcg tagctacaca tgcagtcgaa cggcagcaca ggagagcttg ctctctgggt 60
ggcgagtggc ggacgggtga ggaatacatc ggaatctact ctgtcgtggg ggataacgta 120
gggaaactta cgctaatacc gcatacgacc tacgggtgaa agcaggggat cttcggacct 180
tgcgcgattg aatgagccga tgtcggatta gctagttggc ggggtaaagg cccaccaagg 240
cgacgatccg tagctggtct gagaggatga tcagccacac tggaactgag acacggtcca 300
gactcctacg ggaggcagca gtggggaata ttggacaatg ggcgcaagcc tgatccagcc 360
ataccgcgtg ggtgaagaag gccttcgggt tgtaaagccc ttttgttggg aaagaaatcc 420
agctggctaa tacccggttg ggatgacggt acccaaagaa taagcaccgg ctaacttcgt 480
gccagcagcc gcggtaatac gaagggtgca agcgttactc ggaattactg ggcgtaaagc 540
gtgcgtaggt ggtcgtttaa gtccgttgtg aaagccctgg gctcaacctg ggaactgcag 600
tggatactgg gcgactagag tgtggtagag ggtagcggaa ttcctggtgt agcagtgaaa 660
tgcgtagaga tcaggaggaa catccatggc gaaggcagct acctggacca acactgacac 720
tgaggcacga aagcgtgggg agcaaacagg attagatacc ctggtagtcc acgccctaaa 780
cgatgcgaac tggatgttgg gtgcaatttg gcacgcagta tcgaagctaa cgcgttaagt 840
tcgccgcctg gggagtacgg tcgcaagact gaaactcaaa ggaattgacg ggggcccgca 900
caagcggtgg agtatgtggt ttaattcgat gcaacgcgaa gaaccttacc tggccttgac 960
atgtcgagaa ctttccagag atggatcggt gccttcggga actcgaacac aggtgctgca 1020
tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct 1080
tgtccttagt tgccagcacg taatggtggg aactctaagg agaccgccgg tgacaaaccg 1140
gaggaaggtg gggatgacgt caagtcatca tggcccttac ggccagggct acacacgtac 1200
tacaatggta gggacagagg gctgcaagcc ggcgacggta agccaatccc agaaacccta 1260
tctcagtccg gattggagtc tgcaactcga ctccatgaag tcggaatcgc tagtaatcgc 1320
agatcagcat tgctgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca 1380
tgggagtttg ttgcaccaga agcaggtagc ttaaccttcg ggagggcgct gccacggtgc 1440
cccattgca 1449
Claims (8)
1. The degradation strain for treating the medical wastewater is characterized by being stenotrophomonas parvulaStenotrophomonas pavanii) The strain is named JG-1 and is preserved in China general microbiological culture Collection center (China center for type culture Collection, with the strain number: CGMCC No.24036.
2. A microbial inoculum produced by the degradation strain JG-1 of claim 1.
3. A method for preparing the microbial inoculum of claim 2, comprising the specific steps of:
s1, inoculating a strain JG-1 stored in an inclined plane into an LB solid medium for activation culture;
s2, picking single colonies of the activated strain JG-1, inoculating the single colonies into an LB liquid culture medium, and culturing the single colonies in an oscillating manner until a logarithmic growth phase is reached, wherein the single colonies are used as seed liquid;
s3, inoculating the seed solution into a fresh LB culture medium, continuously performing expansion culture, collecting the culture solution, centrifuging, discarding the supernatant to collect thalli, and finally re-suspending thalli cells with sterile water to obtain the microbial inoculum.
4. The method for preparing a microbial inoculum according to claim 3, wherein in the step S1, the components of the LB solid medium are: 10g/L peptone, 5g/L, naCl g/L yeast extract, 2% agar; the culture conditions are as follows: the temperature is 25-35 ℃ and the time is 24-48 h.
5. The method for preparing a microbial inoculum according to claim 3, wherein in the step S2, the components of the LB liquid medium are: 0.2g/L of p-aminobenzenesulfonamide, 0.2g/L of acetanilide, 10g/L of peptone, 5g/L of yeast extract and 10g/L of NaCl; the culture conditions are as follows: the temperature is 25-35 ℃, the rotating speed is 160-200 r/min, and the time is 12-24 h.
6. The method for preparing a microbial inoculum according to claim 3, wherein in step S3, the fresh LB medium comprises the following components: 10g/L peptone, 5g/L yeast extract and 10g/L NaCl; the culture conditions are as follows: the temperature is 25-35 ℃, the rotating speed is 160-200 r/min, and the time is 24-48 h.
7. The use of the degradation strain according to claim 1 for degrading a pharmaceutical intermediate or a sulfonamide, wherein the pharmaceutical intermediate is one or both of acetanilide and sulfanilamide, and the sulfonamide is one or more of sulfanilamide, sulfapyridine and sulfaisoxazole.
8. The use of the microbial inoculum according to claim 2 in degrading pharmaceutical intermediates or sulfonamides, wherein the pharmaceutical intermediates are one or two of acetanilide and sulfanilamide, and the sulfonamides are one or more of sulfanilamide, sulfapyridine and sulfaisoxazole.
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| CN106282070A (en) * | 2016-10-08 | 2017-01-04 | 南开大学 | The screening of one strain PBDE (PBDEs) degradation bacteria and application |
| CN111690559A (en) * | 2020-06-04 | 2020-09-22 | 江南大学 | Stenotrophomonas maltophilia capable of degrading polyethylene glycol terephthalate |
| CN112788950A (en) * | 2018-07-31 | 2021-05-11 | 微生物公司 | Bismuth-thiol compositions and methods for treating wounds |
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| CN106282070A (en) * | 2016-10-08 | 2017-01-04 | 南开大学 | The screening of one strain PBDE (PBDEs) degradation bacteria and application |
| CN112788950A (en) * | 2018-07-31 | 2021-05-11 | 微生物公司 | Bismuth-thiol compositions and methods for treating wounds |
| CN111690559A (en) * | 2020-06-04 | 2020-09-22 | 江南大学 | Stenotrophomonas maltophilia capable of degrading polyethylene glycol terephthalate |
Non-Patent Citations (2)
| Title |
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| Accelerated biodegradation of polyethylene terephthalate by Thermobifida fusca cutinase mediated by Stenotrophomonas pavanii;Qing-Song Huang et al.,;Science of the Total Environment;第808卷;第1-7页 * |
| 一株可同时降解毒死蜱和联苯菊酯降解菌的筛选鉴定及其降解特性初探;张群 等;农药(第10期);第733-738页 * |
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